A pulse burst optical system has been developed, able to alter an energetic, ultrafast 10 ps, 5 kHz output pulse train to 323 MHz intra-burst frequency at the fundamental 5 kHz repetition rate. An optical delay line consisting of a beam-splitting polariser cube, mirrors, and waveplates transforms a high-energy pulse into a pulse burst, circulating around the delay line. Interestingly, the reflected first pulse and subsequent pulses from the delay line have orthogonal linear polarisations. This fact allows independent modulation of these pulses using two-phase-only Spatial Light Modulators (SLM) when their directors are also aligned orthogonally. With hybrid Computer Generated Holograms (CGH) addressed to the SLMs, we demonstrate simultaneous multi-spot periodic surface micro-structuring on stainless steel with orthogonal linear polarisations and cylindrical vector (CV) beams with Radial and Azimuthal polarisations. Burst processing produces a major change in resulting surface texture due to plasma absorption on the nanosecond time scale; hence the ablation rates on stainless steel with pulse bursts are always lower than 5 kHz processing. By synchronising the scan motion and CGH application, we show simultaneous independent multi-beam real-time processing with pulse bursts having orthogonal linear polarisations. This novel technique extends the flexibility of parallel beam surface micro-structuring with adaptive optics. This limitation has been addressed recently by scaling ultrafast laser output to the multi-hundred Watt level, achieved primarily by increasing repetition rates from tens of MHz to GHz [Ultrashort, femtosecond and picosecond laser pulses are ideally suited for the study of linear light–matter interactions, for example, during pump–probe spectroscopy , non-linz to GHz ,10, althz to GHz . The flez to GHz , which az to GHz ; althougz to GHz  or even z to GHz ,16,17. IAdaptive optics, such as Liquid Crystal on Silicon Spatial Light Modulators (LCOS SLM), can increase the flexibility of light use when combined with ultrafast lasers, accelerating laser micro-fabrication throughput via parallel processing ,19. Thes−2) and average power (p > 200 W) exposures [Incident polarisation is a critical parameter in surface micro-structuring, and Hasegawa and Hayasaki  demonstrxposures ; hence, P > 100 μJ) ultrafast laser pulse into two or more pulses in an optical train with a few nanosecond temporal separation; hence, intraburst frequency νIB > 300 MHz at the fundamental 5 kHz frequency. The pulse bursts include pulse pairs with orthogonal linear polarisations and hence can be modulated independently with two SLMs. This additional degree of control allows a demonstration of dynamic, real-time, near-simultaneous inscription of linear and complex LIPSS with orthogonally polarised pulse bursts at νIB = 323 MHz.The motivation behind the current work is to demonstrate the ability to convert a low-frequency ultrafast laser system from the kHz range to >300 MHz pulse frequency, significantly extending the capability of a low average power ps laser system. The research work here introduces a simple pulse burst optical system able to split a relatively high energy , polarising beam splitter (PBS), dielectric reflecting mirrors, and internal wave plate (HWP2) within the loop. The relative setting of the HWP1,2 fast axes creates reflected and circulating pulses or a pulse burst.A schematic of the optical layout setup is shown in 1 addressed with appropriate CGH and re-imaged via a 4f system (f1 = f2 = 300 mm) to SLM2 where, after further modulation, the beam was again re-imaged  to the input aperture of a scanning galvo system (Nutfield) and focused with a 100 mm focal length f-theta lens to the substrate. The scan software employed was SCAPS GmbH. The beam profile of pulse bursts prior to materials micro-structuring could be observed using a flip mirror and imaged to a Spiricon camera (SP620U) by a long focal length lens . The SLMs are reflective phase-only type, Hamamatsu X13138-5785 (1280 × 1024 pixels) and X10468-03 (800 × 600 pixels), respectively, and SLM2 was mounted with its director vertical, orthogonal to that of SLM1. The CGHs shown are simple hybrid holograms used for beam shaping [1 modulates |H〉 polarised pulses from the delay line while SLM2 modulates only |V〉 polarised reflected pulses from the PBS. Pulse bursts of diffracted horizontal and vertical polarised arrays could be generated with appropriate hybrid CGHs while arrays of orthogonal vector beams [2. Polished 304 stainless steel samples (roughness Ra = 50 nm) were mounted on a three-axis  motion control system (Aerotech) to bring the sample surface to the focal plane.The burst pulses are then incident at low AOI < 10° on SLM shaping  of the q1280 × 10 pixels aor beams  just bey1 rotates the incident linear polarisation at the PBS interface; hence, the amplitude of reflected, |V〉 polarised and transmitted |H〉 polarised components are determined by the HWP1 fast axis setting. The internally transmitted pulse circulates around the delay line, and if no internal waveplate were added, it would exit the delay line at the PBS with |H〉 polarisation, thus creating an orthogonally polarised pulse pair. The addition of HWP2 within the delay line alters internal polarisation so that the circulating pulse is again split into transmitted |H〉 and reflected |V〉 components at the PBS. The relative setting of external and internal waveplates thus allows the creation of a pulse burst with burst number NP ≥ 2 with polarisations, |V〉, |H〉, |H〉, |H〉….and so on. The optical path length from the PBS centre around the loop was measured to be ΔL = 91.7 ± 0.5 cm. Allowing for the PBS dimensions and its refractive index , the effective optical path delay of the loop OPD = 93.0 ± 0.5 cm and hence the burst frequency νIB = 322.8 ± 1.6 MHz determined primarily by the speed of light in air. We designate pulse bursts as  for two pulse orthogonal polarisations with a 3.1 ns delay, while other pulse sequences can be designated  or  for 2-pulse burst orthogonal polarisations (6.2 ns delay) or identical |H〉 polarisations (3.1 ns delay) respectively. Similarly,  represents a 3-pulse burst with the two delayed pulses, |H〉 polarised, orthogonal to the first pulse |V〉 polarised.Referring to IB = 323 MHz intra-burst frequency is shown in A schematic diagram of a 3-pulse burst output at 5 kHz fundamental frequency with ν0 (min)/ω0 (max) where ω0 (min) and ω0 (max) are the measured ablation semi-minor and semi-major axes, close to the 1/e2 radii at intensity focus. The use of a flip-up mirror with a long-focus lens (f = 750 mm) allowed the delay line burst output to be imaged to a Spiricon CCD camera to check relative spot intensities/spatial overlap prior to surface micro-machining experiments. 2, the orthogonal linearly polarised Gaussians were converted to a vector beam array with orthogonal  polarisations and ring intensity distributions, iδ|Az〉 (where δ is a phase angle) are shown in The high purity of the vector beam polarisations was confirmed by adding a wedged beam splitter (B/S) to the CCD camera while rotating the CCD by 90°. As the B/S angle of incidence (AOI) is close to the Brewster angle, it acts as a polarisation analyser. Vector beams with radial/azimuthal polarisations, |Ra〉, |Az〉 and their superpositions, |Ψ〉 = α|Ra〉 + βe± iϕ) and |R〉, |L〉 circular polarisations appear at the poles [xe, ye are unit vectors along the x, y axes, r and φ are cylindrical coordinates where ρ = 2 r2/ω2 and ω is the 1/e2 radius of the Gaussian from which the LG mode can be generated. The intensity distribution reflected from the wedged plate  is then given by (xe ● ye = 0)The double lobes are well-defined, indicating a high vector beam purity. These vector polarisation states can be represented on the equatorial axis of a high-order Poincare sphere (HOPS) while scalar ring modes with pure optical angular momentum OAM E connected to a wide bandwidth digital oscilloscope . p = 100 with pulse energy/spot E ~ 3 μJ (F0 ~ 0.3 Jcm−2); hence, total burst energy on target Ep ~ 12 μJ. While With equal energy  pulse bursts, orthogonally linearly polarised and spatially diffracted, the resulting surface structuring observed on polished stainless steel with a 2 × 2 Gaussian spot array is shown in the SEM (JEOL 7001FEGSEM) images of p = 100/spot with total pulse energy E ~ 7 μJ/spot (F0 ~ 0.26 Jcm−2) and average power <P> = 140 mW exposure at the material surface. The ring mode peak fluence = (1/e) F0 (Gaussian).With the addition of the cylindrically birefringent SWP to the optical line, orthogonal linear polarisations were converted to ring mode Radial and Azimuthal polarisations. This depends only on the direction of the linear polarisations relative to the direction to the SWP axis. Thus, two incident orthogonal linear polarisations from a modulated pulse pair created simultaneous Radial and Azimuthal polarisations. −2 was found to be effective with a 10 ps pulse length.LIPSS are quasi-periodic surface undulations whose source can be regarded as due to the superposition of the incoming wave with a surface scattered wave which affects the spatial energy deposition on the surface ,34. The 0 = 0.3 Jcm−2). Illumination directions are indicated with red arrows. The optical image in E field rotation within the Galvo and LIPSS formation is sensitive to scan direction. When the E field vector is nearly parallel to scan direction, we observe stronger coupling so that low-frequency LIPSS form normal to the major xE component. On the other hand, with E tilted away from the y direction (yE major component) while scanning in x-direction, LIPSS form normal to the E field direction, not that of the yE vector. This can be resolved by rotating scan directions by approximately 20°.Galvo scanning on the surface with orthogonal polarisations allowed fast surface LIPSS texturing. As nematic liquid crystal SLMs can be addressed in real-time, the application of CGHs can be synchronised to the scan motion control system. Two series of CGHs were designed  to demonstrate independent real-time modulation of a two-pulse burst  with near Gaussian spots. A TTL trigger signal from the laser shutter was sent to a NI input/output controller (NI USB 6501), which signalled a Labview environment to synchronously start the CGH application to the two SLMs. The shutter was opened for 20 ms (50 pulses) and closed for 980 ms every second, which matched the CGH frequency, set to 1.0 Hz. Hence, we demonstrate the synchronous application of CGHs to both SLMs while independently modulating their relative spatial separations during a pulse burst. p = 150 pulses, Ep = 4 μJ/pulse (F0 = 0.4 Jcm−2) is shown in the SEM image, IB = 323 MHz with the same fluence/pulse (Np = 150 pulses) demonstrates a major difference in surface texture with evidence of melting and surface smoothing, Ablation characteristics of pulse burst processing compared to low-frequency (kHz-MHz) have been shown to vary significantly in the literature ,16,17. W2O3 and Fe3O4, which have enthalpies of 824 kJ/mol and 1118 kJ/mol, respectively [ectively . Other e0 = 0.4 Jcm−2, scan speed s = 40 mm/s, and hatch distance = 20 μm. The residual surface micro-structure for 5 kHz  exposure shows clear LIPSS, We machined stainless steel in a square pattern, 0.4 mm dimension and 20 μm deep with 5 kHz,  and  exposure, with 2-pulse bursts ,  and 3-pulse bursts , respectively. Pulse fluence was F−2. Ablation depth increased linearly with pulse number in every case, but we found volume ablation rates decreasing as follows: R ~ R > R > R > R with ablation volume/pulse = 6.9 μm3/pulse, 6.7μm3/pulse, 4.6 μm3/pulse, 3.2 μm3/pulse, 2.7 μm3/pulse, respectively. The ablation rate of the  pulse burst is, therefore, only 39% of that at 5 kHz. Interestingly, the ablation volume/pulse of the  pulse burst (3.1 ns delay) is 30% lower than that of the  burst with a 6.2 ns delay. These observations strongly support plasma absorption as the source of the decreasing ablation rate with pulse bursts and are in agreement with the experimental results of other authors [Ablation rates were estimated from the micro-machined squares, 0.4 mm × 0.4 mm × 20 μm deep at fluence F = 0.4 Jcm authors ,15. Whil0 = 5 kHz) to a pulse burst with intra-burst frequency νIB = 323 MHz or 162 MHz. Up to 3 pulses with equal energy could be generated, allowing the study of the pulse burst laser–materials interactions and comparison with kHz processing. As the pulses reflected from the PBS were vertically |V〉 polarised while transmitted components were horizontally |H〉 polarised, this fact allowed independent polarisation modulation and diffraction of the pulse bursts with the aid of two phase-only SLMs whose directors were also orthogonally aligned.There is currently great interest in pulse burst processing of materials with ultrafast lasers . Here, wBy employing hybrid CGHs on each CGH, beam shaping of the elliptical laser mode was achieved, altering eccentricity from ε = 0.72 to ε = 0.88; hence, spot roundness was improved significantly, although at the cost of lower transmission efficiency. Consequently, pulse burst arrays of spots with orthogonal linear polarisations were modulated simultaneously, imprinting surface LIPSS with orthogonal directions. By introducing a nano-structured SWP into the optical line, CV beam arrays with ring modes and orthogonal polarisations  simultaneously imprinted more complex low-frequency LIPSS, also confirming the high purity of these interesting polarisation states, regarded as classically non-separable in spatial and polarisation modes ,42. As nA comparison of 5 kHz multi-pulse  and 3-pulse burst processing  at 323 MHz intraburst frequency on fixed spots demonstrated a remarkable change in surface texturing of stainless steel during blind drilling. While low-frequency LIPSS are characteristic of multi-pulse ultrafast kHz ablation at fluences just above the ablation threshold, pulse bursts with nanosecond delays introduced significant surface melting due to plasma absorption, which, using the pump–probe technique, can be maximum after a few ns delay . With puWith an optical setup involving two SLMs, a 4f optical system, and a delay line burst generator, it is worth reporting the overall efficiency of laser light used at 5 kHz compared to a pulse burst with and without beam shaping. With the , 5 kHz output and no beam shaping, the overall transmission efficiency from the PBS to the Galvo input aperture was measured to be η = 63%, while with the hybrid CGHs applied to both SLMs, this dropped to η = 50%. With the  two-pulse burst without beam shaping, η = 61%, while with hybrid CGHs applied, efficiency dropped to η = 43%. These results compare favourably with those measured by Hasegawa and Hyasaki  with twoVery recently, a delay line consisting of a non-polarising beam splitter  and three mirrors was used to create pulse bursts, all with the same polarisation at 1 GHz intra-burst frequency for fs laser processing of steel and copper . Bursts The work presented here also demonstrates dynamic polarisation and spatial modulation of orthogonally polarised pulse bursts with 2 reflective SLMs, increasing the flexibility of laser processing of materials. If the output laser beam were truly Gaussian, the losses incurred by beam shaping would be reduced, hence, increasing light use efficiency to η ~ 63%, although this will be CGH dependent. The technique developed in this work would allow simultaneous processing at kHz repetition rates combined with pulse burst processing, dependent only on CGH application, and might well be applied in the spatial control of surface materials wettability, surface bio-compatibility, or encoding complex surface structures for security marking of valuable components. In addition, it may be possible to directly write high-quality reflective optical gratings or plasmonic sensors into metallic surfaces. By optimising the synchronisation scheme, orthogonal burst polarisation modulation at frequencies >10 Hz should be possible, limited only by the SLM bandwidths <30 Hz . With cu2 flat scan field, while the translation stages have a 100 mm range. By combining these motions, the processing area can be increased to approximately 130 × 130 mm2, industrially relevant. The novel optical system developed expands the current window utilising techniques available for high throughput, dynamic ultrafast laser processing, particularly when combined with kHz, high energy, high average power systems currently under development [An optical pulse burst optical system operating at intra pulse burst frequency of 323 MHz from a 5 kHz fundamental frequency has been developed to study the differences in ultrafast laser–material processing of stainless steel. At 5 kHz, micro-structuring produces clear low-frequency LIPSS both for fixed spots and during scanning when the beam overlap is approximately 33%. On metals, these low-frequency LIPSS, with period Λ ~ λ (laser wavelength), appear near right angles to the local E vector. On the other hand, pulse bursts with 3.1 ns between pulses lead to plasma absorption, which re-heats the surface, leading to melting, so that LIPSS disappear. As a two-pulse  burst has orthogonal polarisations, each pulse can be modulated independently by an SLM with directors oriented appropriately. This has allowed independent, real-time, two-beam surface processing with orthogonal and complex LIPSS. The current Galvo system with a 100 mm focal length f-theta lens has a 60 × 60 mmelopment .


Moreover, SPVPC exhibited excellent adsorption characteristics for removing methylene blue (MB) from aqueous solutions. The maximum adsorption capacity for MB reached 653.6 mg g−1, and the reusability was satisfactory. The adsorption kinetics and isotherm were in good agreement with the pseudo-second-order kinetics and Langmuir models, respectively. The adsorption mechanism was summarised as the synergistic effects of the hierarchically porous structures in SPVPC and various interactions between SPVPC and MB. Considering its low cost and excellent adsorption performance, the prepared porous carbon is a promising adsorbent candidate for dye wastewater treatment.In this study, sweet-potato-vine-based porous carbon (SPVPC) was prepared using zinc chloride as an activating and pore-forming agent. The optimised SPVPC exhibited abundant porous structures with a high specific surface area of 1397.8 m Dye wastewater has attracted wide attention, with approximately 100 tons of dye discharged into natural water bodies annually, causing serious water pollution and ecosystem destruction . It poseCurrently available techniques, such as membrane separation, biodegradation, adsorption, electrochemical, photocatalysis, oxidation, and flocculation, have been employed to purify dye wastewater . In contIpomoea batatas L.) is planted worldwide, and its tuberous root is usually used as a food source [2) as an activating and pore-forming agent, and its structural characteristics and adsorption performance towards MB were analysed in detail.Sweet potato  and time (t), were investigated using a single factor methodology to obtain high-performance SPVPC. As seen in qe increased sharply with the increasing MR until 2:1, with the highest value being 299.6 mg g−1, but it slightly decreased when the MR was over 2:1. Moreover, the effect of T on qe showed a similar pattern and qe reached a maximum value of 299.2 mg g−1 at 500 °C  [The surface morphology of the optimised SPVPC was observed using SEM. As shown in esopores . Meanwhiolecules . Surprisrted PCs ,23,24,25 [2 g−1) .−1 (D-band) is generally attributed to disordered and defective carbons with the breathing mode of the A1g symmetry, and 1595 cm−1 (G-band) corresponds to ordered graphitic structures with the zone centre of the E2g mode [−1, 2926 cm−1 and 2860 cm−1, 1697 cm−1, 1568 cm−1, 1165 cm−1 and 1070 cm−1, 889 cm−1 and 812 cm−1, ascribed to the stretching vibrations of O–H (hydroxyl), C–H (methyl and methylene), C=O (carbonyl and carboxylic), C=C (benzene ring), C–O , and C–H (benzene ring), respectively [The crystallographic characteristics of the SPVPC were explored using the XRD pattern shown in E2g mode ,26. FurtE2g mode ,19. Addiectively ,31. Therectively . Howeverectively . Importaectively .qe gradually increased from 218.8 mg g−1 to 299.4 mg g−1. MB is a cationic dye with a positive charge in an aqueous solution [zero charge point, qe increased sharply owing to the reinforced electrostatic interaction between SPVPC and MB. When pH < 4.1, electrostatic repulsion between the positively charged SPVPC surface and MB occurred [+ competed with MB for the surface adsorption sites of SPVPC, leading to a lower qe [qe could reach 218.8 mg g−1 at pH 2, indicating that adsorption was regulated by electrostatic interaction and other interactions, such as π–π attraction, and hydrogen bonding between SPVPC and MB molecules. Moreover, qe increased with increasing c0  within 1 min , and its calculated qe,cal was consistent with the experimental qe,exp, revealing that PSO was more suitable for explaining the adsorption process, and chemisorption mainly dominated the adsorption behaviour of SPVPC towards MB [R2 (0.9995) of Langmuir was much higher than that of Freundlich (0.8423), suggesting that the adsorption process fit the Langmuir isotherm and a monolayer coverage of MB molecules occurred on the SPVPC surface, which was consistent with previously reported PCs [qm of 653.6 mg g−1 was higher than that of most reported adsorbents , sodium hydroxide (NaOH), and other analytical reagents were purchased from the Chengdu Kelong Chemical Company .Sweet potato vines were collected from a sweet potato field in Yongchuan , dried, milled, and passed through a 60 mesh sieve to obtain a powder with a particle size ≤0.25 mm for the carbonisation experiments. MB was purchased from the Shanghai Aladdin Chemical Company . ZnCl2 activation method. Typically, 1.0 g of SPV powder and 0.5–4 g of ZnCl2 were dispersed in 50 mL of water with vigorous magnetic stirring for 1 h. The mixtures were then dried using a vacuum freeze dryer. The dried samples were heated and carbonised in a tube furnace at 300–700 °C for 0.5–2.5 h at a heating rate of 5 °C min−1 under a nitrogen flow. After natural cooling, the obtained samples were ground and washed with a HCl solution (1 mol L−1) and ultrapure water to remove inorganic impurities [SPVPC was prepared using the ZnClpurities ,20. Fina−1. The microcrystalline structure of graphite was characterised using a Renishaw inVia plus micro-Raman spectroscopy system . The surface groups were recorded on a Nicolet 6700 Fourier transform infrared spectrophotometer .The morphology was observed using a JSM-7800F scanning electron microscope  at 10 kV. The specific surface area and pore characteristics were determined using a porosity analyser . The crystalline structure was determined using an Ultima IV X-ray diffractometer  with Cu-Kα radiation (λ = 0.15 nm) and a scan speed of 4° min−1), pH values (2–12), times (1–120 min), and temperatures (25–50 °C) in an incubation shaker at a speed of 200 rpm. The mixtures were then centrifuged, and their supernatant absorbances were measured using a TU-1901 UV–vis spectrophotometer  at 664 nm. The final concentrations of MB were determined using the standard curve of absorbance and concentration. The formulas of the adsorption capacity, pseudo-first-order (PFO) and pseudo-second-order (PSO) kinetics, and Langmuir and Freundlich isotherms are as follows.qe (mg g−1) and qt (mg g−1) are the adsorption capacities of SPVPC toward MB at equilibrium and any time t (min), respectively; c0 (mg L−1) and ce (mg L−1) are the initial and equilibrium concentrations of MB, respectively; V (L) is the MB solution volume; m (g) is the SPVPC mass; k1 (min−1) and k2 (g mg−1 min−1) are the rate constants of the PFO and PSO kinetics, respectively; qm (mg g−1) is the max adsorption capacity; b (L mg−1) and k ((mg g−1(L mg−1)1/n) are the constants of the Langmuir and Freundlich isotherms, respectively; and 1/n is the heterogeneity factor of the Freundlich isotherm.Batch adsorption experiments were performed by adding 10 mg SPVPC into 10 mL of MB aqueous solutions with different initial concentrations . After adsorption, the MB-adsorbed SPVPC was separated by centrifugation, and 10 mL of ethanol (pH = 2) was added for desorption through ultrasound treatment for 5 min. Finally, the desorbed SPVPC was isolated by centrifugation for the next cycle. This process was repeated five times.Typically, 10 mg of SPVPC was mixed with 10 mL of a 100 mg L2 activation strategy. The obtained SPVPC possessed a high specific surface area, rich pores, strong adsorption ability, and excellent reusability for MB removal. The adsorption process fit pseudo-second-order kinetics and Langmuir isotherm models. The adsorption mechanism was considered to be the integrated effect of hierarchical pores in SPVPC and the various interactions between SPVPC and MB molecules. This study provides a novel and promising absorbent material for treating dye wastewater. Prospectively, the microorganism and enzyme are possible alternatives and ecofriendly porogens to treat the biomass precursor for preparing PC.In this study, SPVPC was successfully prepared using a facile ZnCl


We can, hence, conclude that the enhanced burden of threat appraisals observed after SARS-CoV-2 spread leads the way to establish preventive, diagnostic and therapeutic measures in order to limit the unfavorable mental health consequences caused by the ongoing COVID-19 pandemic.We planned an infodemiological analysis to estimate the global impact of coronavirus disease 2019 (COVID-19) on threat appraisals. We accessed Google Trends using the search terms “Anxiety”, “Distress”, “Fear”, “Rumination”, “Stress” and “Worry” within the “topic” domain, setting the geographical location to “worldwide”, between July 2017 and July 2022. The weekly Google Trends score for the six search terms, thus, mirroring Web popularity and probable prevalence, was compared between the two search periods, “pre-COVID” (between July 2017 and February 2020) and COVID (between March 2020 and July 2022), thus, reflecting the volume of searches before and during the ongoing COVID-19 pandemic. The median weekly Google Trends score of all these search terms significantly increased during the COVID-19 pandemic, i.e., anxiety by 22%, distress by 13%, fear by 9%, rumination by 18%, stress by 13% and worry by 20%. With variable strength, the weekly Google Trends scores of each search term were found to be significantly associated (all  Coronavirus disease 2019 (COVID-19), first identified at the end of 2019 in the Chinese city of Wuhan , may cauIn addition to the risk of developing severe and occasionally persistent organic derangements, the ongoing COVID-19 pandemic is also apparently enhancing the threat appraisals of both affected and unaffected individuals. Several recent studies, involving populations of all ages, from different geographical locations and not necessarily engaged in patient care, have, in fact, reported an enhanced burden of psychological disturbances, including anxiety and stress, namely due to concern/fear of being infected and developing acute and/or long-term unfavorable consequences of COVID-19 ,6,7,8. IWe accessed Google Trends  using the search terms “Anxiety”, “Distress”, “Fear”, “Rumination”, “Stress” and “Worry” on 1 August 2022, within the “topic” domain , setting the geographical location to “worldwide” and limiting our analysis to the past 5 years . These search terms were selected as being largely used in previous publications to define unfavorable post-COVID-19 mental health outcomes ,7,8. TheThe study was conducted in accordance with the Declaration of Helsinki, under the terms of relevant local legislation. This analysis was based on electronic searches in an open and publicly available repository (Google Trends) and, thus, no informed consent or Ethical Committee approval were necessary.The results of this infodemiological analysis are shown in p < 0.001), distress by 13% from 60  to 68 , fear by 9% from 73  to 80 , rumination by 18% from 57  to 67 , stress by 13% from 76  to 86 , and worry by 20% from 64  to 77 . The Spearman’s correlation between the median weekly Google Trends score of all these search terms over time is summarized in The median weekly Google Trends score of all the investigated domains significantly increased during the COVID-19 pandemic. Specifically, anxiety increased by 22% from 64  to 78 , with the highest correlation found between the Google search volumes of stress and worry (r = 0.81), followed by anxiety and rumination (r = 0.80), anxiety and worry (r = 0.80), distress and worry (r = 0.80). The weakest correlation was found between the Google Trends scores of fear and rumination (r = 0.57), as well as those of fear and stress (r = 0.62).With variable strength, the weekly Google Trends scores of each search term were found to be significantly associated (all Important evidence has emerged from several clinical trials, recently meta-analyzed by Lekagul and colleagues , revealiAlthough no sub-analysis could be carried out since the searches in Google Trends cannot be more specifically driven to cluster demographical information, our findings reveal that COVID-19 may have imposed a considerable mental health impact all around the world, as attested by the significantly increased volume of worldwide Google searches for terms, such as “Anxiety”, “Distress”, “Fear”, “Rumination”, “Stress” and “Worry”, after March 2020, when the WHO declared COVID-19 a pandemic  15]. Mo. Mo15]. The evidence that the threat appraisals may have dramatically increased after the spread of SARS-CoV-2 calls for an urgent need to establish preventive, diagnostic and therapeutic measures, such as those previously mentioned, in order to limit the unfavorable mental health consequences caused by the ongoing COVID-19 pandemic.


Previous research has demonstrated that chronic diseases can occur due to musculoskeletal (MS) pain and poor sleep. It is also worth noting that the caffeine in coffee can reduce overall sleep duration, efficiency, and quality. Thus, the present study examines the effects of frequent coffee drinking (two cups per day) on individuals experiencing MS pain and a lack of sleep during the COVID-19 period. This observational and cross-sectional study recruited 1615 individuals who completed the self-reported  questionnaire. Long-term, frequent coffee drinking and a sleep duration of less than 6 h per day were significantly associated with neck and shoulder pain among healthy individuals. The mediation model demonstrated that the shorter sleep duration and drinking multiple cups of coffee per day had a two-way relationship that worsened such pain over the long term. Specifically, individuals who experienced such pain frequently drank multiple cups of coffee per day, which, in turn, shortened their sleep durations. In summary, long-term coffee drinking creates a vicious cycle between MS pain and sleep duration. Therefore, the amount of coffee should be fewer than two cups per day for individuals who sleep less than 6 h per day or suffer from MS pain, especially neck and shoulder pain. Coffee is one of the most popular beverages for people of many ages. It is a complex chemical mixture that contains caffeine, which is a purine alkaloid that is naturally found in coffee beans  and contMS pain is common in many occupations, and it is one of the main reasons for long-term sick leave . In the Poor sleep quality is a common health problem among medical staff ,29. ReduOverall, a close relationship was found between sleep and MS pain. For instance, because sleep problems can significantly reduce pain tolerance , individFrom a micro and physiological perspective, adenosine is a purine nucleoside and a ubiquitous endogenous neurotransmitter that signals through four receptors  in the brain to inhibit arousal and increase drowsiness . Among tHypothesis 1:Coffee intake is significantly associated with increased risk of MS pain.Hypothesis 2:Individuals with shorter sleep durations are more susceptible to MS pain.Hypothesis 3:Coffee intake could lead to a vicious circle between lack of sleep and MS pain.This observational and cross-sectional study was initially conducted from a hospital affiliated with a medical university in Taichung, Taiwan, from March to April 2021. All 2531 healthcare workers who had served for one year in the hospital were distributed a QR code for a Google Forms-linked questionnaire by email. Among them, 1633 (64.52%) individuals completed the self-reported questionnaire, after which 1615 (63.81%) were deemed valid after those with missing data were excluded. Specifically, we used questionnaires, including the Nordic musculoskeletal questionnaire (NMQ), to obtain the participants’ basic demographic variables, family factors, living habits, work, physical health, and MS pain. The study protocol was approved by the Institutional Review Board of Chung Shan Medical University Hospital on 25 August 2021 (No: CS1-21108).This study adopted the NMQ, modified and translated by the Taiwan Institute of Occupational Safety and Health , to survIn the questionnaire, the basic response options included male or female for gender; age; “married” or “other” for marriage; and “without child,” “one child,” “two children,” “three children,” and “more than three children” for having children. The survey also asked if the participants engaged in leisure activities with family/friends during vacation time. The response options included “always,” “often,” “sometimes,” “seldom,” and “never.” Regarding their education, the response options were “master’s degree or above” and “university degree or below,” while the response options for self-reported sleep duration per day included “less than 5 h,” “between 5 and 6 h,” “between 6 and 7 h,” “between 7 and 8 h,” and “more than 8 h.” As for their coffee intake per day, the response options were “more than 2 cups per day,” “2 cups per day,” “1 cup per day,” “occasionally,” and “never.” Regarding their alcohol use, the response options included “alcohol use in a month” and “no alcohol use in a month,” while the response options were “yes” and “no” for exercising at least once a week. As for their overtime work, the response options were “seldom,” “fewer than 45 h per month,” “45 to 80 h per month,” and “more than 80 h per month,” while “irregular,” “regular,” “night,” and “day” were the response options for shift schedules. Finally, the participants were classified as physicians, nurses, professional and technical personnel, and administrative staff. They were also asked about the presence of chronic diseases. In this regard, the presence of one or more diseases was classified as a “yes” response.t-test or one-way ANOVA was adopted to examine the differences between the continuous variables. Additionally, a chi-square test or Fisher’s exact test was conducted to determine the significant differences in the categorical variables, while simple/multiple linear or logistic regression was used to examine the correlation between the dependent variable (DV) and the independent variable (IV), in the absence (or presence) of the controlled variables. The mediation effects among the IV, DV, and mediator were based on the following strategy proposed by Baron and Kenny [Regarding the statistical methods, factor analysis  was adopnd Kenny : 1) in tA mediation model suitable for combining the categorical and continuous variables was developed by Iacobucci (2012) . The forIf the mediation factor and dependent variables are continuous variables, then the original formula of the Sobel test is applicable:If the mediation factor or dependent variables are categorical variables, then the original formula of the Sobel test is rederived into a new formula:p < 0.05.Among them, p = 0.016), engaging in leisure activities with family/friends (p < 0.0001), coffee intake per day (p < 0.0001), exercise at least once a week (p = 0.008), overtime work in a month (p < 0.0001), shift schedules (p < 0.0001), and profession (p = 0.005) were associated with sleep duration per day. In addition, gender (p = 0.024), age (p < 0.0001), marriage (p < 0.0001), having children (p < 0.0001), education (p < 0.0001), alcohol use (p < 0.0001), exercise at least once a week (p = 0.002), and profession (p = 0.001) were related to coffee intake.Regarding the detailed description, the description of the basic demographics, sleep duration per day, and coffee intake of 1615 participants are shown in the p < 0.001), age (p = 0.003), marriage (p = 0.003), having children (p = 0.006), education (p = 0.034), sleep duration per day (p < 0.001), coffee intake per day (p = < 0.001), alcohol use (p = 0.001), exercise at least once a week (p = 0.001), overtime work per month (p < 0.0001), profession (p = 0.036), and suffering from chronic diseases (p < 0.0001). There were no significant differences in the BAP scores among the survey variables, except for education (p < 0.0001). Regarding the other survey variables, the females obtained higher NBSP scores than the males (0.04 ± 0.93 vs. −0.17 ± 0.84). Moreover, individuals who were 38–45 years of age (0.15 ± 0.96), were married (0.07 ± 0.96), were parents (0.07 ± 0.96), had a master’s degree or above (0.11 ± 0.99), had a sleep duration of less than 5 h (0.26 ± 1.04), drank more than two cups of coffee per day (0.61 ± 1.25), used alcohol in a month (0.10 ± 0.97), had no weekly exercise (0.09 ± 0.97), worked overtime more than 45 h per month (0.54 ± 1.35/0.44 ± 1.14), were nurses (0.08 ± 0.94), or suffered from chronic diseases (0.20 ± 1.03) achieved higher NBSP scores than the others. Simple multiple linear or logistic regression was also used to examine the correlation between the dependent and independent variables in the absence (or presence) of the controlled variables.According to Since the number of individuals with a sleep duration of less than 5 h per day was only 63 , the varp = 0.001; 0.23, p = 0.016), while SLD < 6 h was significantly associated with an increased level of NBSP in the simple or multiple linear regression models . The results in As shown in m = 2.27, p < 0.05). Lack of sleep (<6 h) also caused individuals to drink more coffee per day, which led to more frequent neck and shoulder pain. m = 2.95, p < 0.01). Overall, Finally, this study used mediation analysis to determine the existence of a mutual relationship between coffee, MS pain, and sleep. m = 2.5, p < 0.05). Specifically, individuals who suffer from neck and should pain tend to drink coffee to cope with such pain. However, it eventually decreases their sleep duration per day. The present study confirms three hypotheses and determined that long-term heavy coffee intake (two cups per day) and a shorter sleep duration (<6 h per day) are associated with neck and shoulder pain. Notably, long-term heavy coffee intake plays a mediating factor in the vicious circle between shorter sleep duration and neck and shoulder pain. In addition, alcohol use, the lack of regular exercise at least once a week, overtime work in a month, and the presence of chronic diseases were significantly associated with pain in the neck/shoulders or ankles.Related studies have illustrated that reduced alcohol use ,19,20,21A literature review on healthcare workers demonstrated that MS pain occurred primarily in the lower and upper back, neck, and shoulders . Our stuOnly 25% of adults achieve the recommended minimum sleep duration of 7 h per night for healthy adults . Howeverp = 0.016). Since the half-life of caffeine is approximately 4 h [Previous studies have found that individuals with a high caffeine intake (4–12 cups/day) had more severe pain than those with a low (0.25–1.5 cups/day) or moderate (2–3.5 cups/day) caffeine intake . In additely 4 h , drinkintely 4 h . Based op = 0.001). These results confirm our second hypothesis.Evidence suggests a close link between short sleep durations and impairments in several physiological responses, including pain . A studyp < 0.0001) more caffeine per day than those who reported a sleep duration of >6 h. This close relationship between sleep duration and coffee intake indicates a causal relationship between sleep duration, NBSP, and coffee intake.A previous study showed that individuals who reported a sleep duration of <6 h consumed 3.6 times more caffeine per day than those who reported a sleep duration of >8 h . Our stup < 0.05). In addition, the mediation model in p < 0.01). These mediation models regarding coffee intake, MS pain, and sleep duration show that long-term heavy coffee intake (more than two cups per day) plays a mediating role in the two-way association of sleep duration <6 h and NBSP. Specifically, long-term heavy coffee intake will induce a vicious circle of sleep and neck and shoulder pain. These results are consistent with our third hypothesis.The mediation model in This study has several limitations. First, we used the number of cups to measure the degree of caffeine intake per day. However, this is not an exact measurement method because cups have different volumes. Second, different coffee-brewing methods can lead to varied caffeine concentrations and errors in the dose–response of caffeine on MS pain. However, we believe that the differences in volume and caffeine concentration can be overcome. In addition, caffeine’s effect on MS pain reaches the threshold depending on one’s genetics . TherefoDespite the adjustment for sex in the multiple linear regression, the results of the present study could be better suited to women because female participants accounted for >80% of the study population. Regarding the sex difference in MS pain, it could be caused by estrogen and progesterone. For instance, testosterone, the major male sex hormone, protects men from chronic MS pain . BecauseNotably, we could determine whether high work stress or emotional exhaustion caused by the pandemic affected the findings; thus, a similar study during the nonpandemic period should be replicated, and its results compared with those from the pandemic period. Finally, the mediation models in our study could be biased  because The present study determines that keeping good living habits , maintaining physical health (such as staying away from chronic diseases), and avoiding overtime work are ways to lower the risk of MS pain. We further examined the effects of frequent coffee drinking on individuals experiencing MS pain and lack of sleep. Based on the results, neck and shoulder pain was the most common among the healthcare workers. In addition, a sleep duration of less than 6 h and drinking more than two cups of coffee per day increased the occurrence of such pain, while controlling for other risk factors. Notably, long-term heavy coffee drinking created a vicious cycle between neck and shoulder pain and sleep duration of less than 6 h. The implication of the findings is that individuals who sleep less than 6 h, or who suffer from neck and shoulder pain, should limit their coffee intake to two cups per day.


Heavy metal (HM) contaminated soil can affect human health via ingestion of foodstuffs, inhalation of soil dust, and skin contact of soil. This study estimates the level of some heavy metals in soils of industrial areas, and their exposures to human body via dietary intake of vegetables and other pathways. Mean concentrations of Cr, Fe, Cu, Zn, As and Pb in the studied soil were found to be 61.27, 27,274, 42.36, 9.77, 28.08 and 13.69 mg/kg, respectively, while in vegetables the respective values were 0.53, 119.59, 9.76, 7.14, 1.34 and 2.69 mg/kg. Multivariate statistical analysis revealed that Fe, Cu, Zn, and Pb originated from lithogenic sources, while Cr and As are derived from anthropogenic sources. A moderate enrichment was noted by Cr, As, and Pb in the entire sampling site, indicating a progressive depletion of soil quality. The bioaccumulation factor (BCF) value for all the vegetables was recorded as BCF < 1; however, the metal pollution index (MPI) stipulates moderately high value of heavy metal accumulation in the vegetable samples. Hazard Index (HI) of >0.1 was estimated for adults but >1 for children by direct soil exposure, whereas HI < 1 for both children and adults via dietary intake of vegetables. Estimated Total carcinogenic risk (TCR) value due to soil exposure showed safe for adults but unsafe for children, while both the population groups were found to be safe via food consumption. Children are found more vulnerable receptors than adults, and health risks (carcinogenic and non-carcinogenic) via direct soil exposure proved unsafe. Overall, this study can be used as a reference for similar types of studies to evaluate heavy metal contaminated soil impact on the population of Bangladesh and other countries as well. The fundamental part of the ecosystem is the soil which supplies necessary nutrients to living organisms. Soil receives different types of metals  from various sources (anthropogenic and lithogenic), however, an increase of their natural content may reduce the soil quality. In fact, as a reservoir, the soil itself is abundant in metals transported from biomass, atmosphere, and hydrosphere ,2, but iHeavy metal (HM) contaminated soil may pose potential risks and hazards to humans by direct ingestion or contact with contaminated soil or inhalation of contaminated soil dust and intake of foodstuffs. Thus, health risks arising from soil can be estimated by calculating various soil pollution indices of HMs, their soil-to-plant transfer factors, direct exposure level to humans, and their levels in edible food crops as well as health risk due to consumption of contaminated food crops. Numerous studies have been conducted all over the world and so as in Bangladesh, to estimate the health hazards of heavy metal contaminated soil ,11,12,13Ashulia, a neighbouring community of Dhaka district , which is a suburban area, and Savar a nearby area having the same soil texture were targeted as the sampling area. A vast number of paddy fields and agricultural lands are located in this area. Ashulia Lake and two major theme parks in Bangladesh, namely “Fantasy Kingdom” and “Nandan Park”, make it a popular tourist location. A huge number of local and foreign tourists including children visited this area frequently. However, in recent years, rapid urbanization, the establishment of garments factories, bricks fields and other factories deteriorated its beauty and reduced the farmlands. Frequent dumping of untreated solid and liquid wastes from the factories and brickfields to the nearby agricultural land makes them assailable due to the risk of metal accumulation into crops, vegetables and ultimately the human body. As far as our concern, no studies have been conducted to monitor this industrially affected land soil, crops grown in these soils and their impact on humans. In this context, the present study was aimed to measure some essential and non-essential metals  in soil contaminated by industrial wastes, and vegetables grown on this soil, to determine the degree of pollution by estimating various indices, and to find out the possible pollution sources by employing multivariate statistical analysis. Health risk owing to soil-human and the soil-plant-human route was assessed and a comparison was made to ascertain which path is more vulnerable. Translocation of heavy metals from soil to edible parts of vegetables was also carried out to find out the metal extraction capability of the plants from a phytoremediation point of view., where industrial input was observed to be absent. To evaluate contaminated soil impact on human health via food consumption, 15 varieties of vegetables namely Spinach, Cabbage, Red Amaranth, Coriander leaf, Tomato, Brinjal, Bean, Pumpkin, Bottle gourd, Papaya, Green banana, Cauliflower, Carrot, Radish, Potato was collected that have been grown in and around the industrially affected soil sampling sites , IS2 (23°54′19.9″ N 90°17′36.3″ E), IS3 (23°54′16.3″ N 90°17′38.2″ E), IS4 (23°54′15.9″ N 90°17′38.4″ E), IS5 (23°54′16.1″ N 90°17′39.8″ E), IS6 (23°54′12.4″ N 90°17′37.3″ E), IS7 (23°54′12.9″ N 90°17′18.0″ E), IS8 (23°54′35.6″ N 90°17′31.8″ E), IS9 (23°54′42.6″ N 90°17′31.1″ E) and IS10 (23°54′47.0″ N 90°17′30.3″ E) were collected from the upper surface region (5–15 cm depth) of agricultural land of Ashulia, Dhaka . A largeng sites .Each soil sample was dried to remove moisture, ground to a fine powder and finally sieved to obtain homogeneous particles. Vegetable samples were initially cleaned with tap water and rinsed with deionized water to remove any trace of soil and to minimize ion content, cut into small pieces, and dried to remove moisture. The dried mass was ground to a fine powder. Finally, 0.1 gm of each soil and vegetable sample in triplicate were pressed into a pellet of 0.7 cm diameter and 1 mm thickness using a pellet maker. The whole process is outlined by following the procedure described elsewhere ,22.Soil and vegetable samples, each in triplicates, were analysed for heavy metals  using Energy Dispersive X-ray Fluorescence (EDXRF) system. It’s a non-destructive and multi-elemental analytical technique, well suited for solid sample  analysis. Many research works have been conducted with this technique for heavy/trace metal determination in environmental and food samples ,17,22,23geo), Contamination factor (CF), and Pollution load index (PLI) as per [sample refers to the ratio of concentration between the studied metal and Fe in the sample of interest; (Me/Fe)background is the natural background value (control soil in this case) of measured metal to Fe ratio [geo is the geo-accumulation index; Cn is the individual heavy metal concentration; Bn is the geochemical background value  and factor 1.5 is introduced to include possible variations of the background values due to the lithogenic effect [geo value can be categorised [geo ≤ 0 (unpolluted), Igeo = 0–1 (unpolluted to moderately polluted), Igeo = 1–2 (moderately polluted), Igeo = 2–3 (moderately to strongly polluted), Igeo = 3–4 (strongly polluted), Igeo = 4–5 (strongly to extremely polluted) and Igeo = 5–6 (extremely polluted).samplem is the concentration of a given metal; Cbackgroundm is the background value of the metal (control soil) [The degree of soil pollution was measured by calculating the Enrichment factor (EF), Geo-accumulation index (I) as per , whereas) as per ,23. The Fe ratio . Howeverc effect . Igeo vaegorised  as Igeo ol soil) . CF is col soil)  as CF < rised by  as PLI <Multivariate statistical methods are usually applied to evaluate the complex eco-toxicological processes regarding the relationship and interdependency among the variables and their relative weights . In thisThe data obtained from this study were analysed statistically using SPSS version 25.0 software , graphs were displayed using Microsoft Excel 2019, and Box-whisker plots were plotted with Origin Pro software version 9.0. The analysis of variance (ANOVA) tests at a significance level of 95% were used to evaluate the impact of different variables on the contamination in the study area. PCA and CA were performed using SPSS.Vegetable contamination levels were calculated by using Bioaccumulation Factor (BCFs) and Metal pollution index (MPI) as per ,23.veg is the concentration of heavy metal in the vegetable , and Csoil is the concentration of heavy metal in the soil  [The Equation (5) is used to calculate bioaccumulation factors (BCFs) of the heavy metals from soil to plant,/kg, dw) . It is n/kg, dw) .n is the concentration of n number of metals in the sample.The Metal Pollution Index (MPI) index was obtained by calculating the geometrical mean concentration of all the metals in the analysed vegetable samples ,34.(6)MHeavy metal contaminated soil can affect human health in two pathways: (1) soil to humans via direct soil (dust) exposure; (2) soil to food to humans via consumption of foodstuffs.ing); inhalation (ADDinh); dermal absorption of metals via the skin  [inges + ADDinha + ADDderm) and RfD refers to the heavy metal (HM) oral reference dose. RfD for ingestion: Fe = 7.00 × 10−1, Cr = 3.00 × 10−3, Cu = 0.04, Zn = 0.3, As =3.00 × 10−4 and Pb = 0.0035; for inhalation: Cr =2.86 × 10−5, Cu = 0.0402, Zn = 0.3, As = 3.01 × 10−4 and Pb = 0.00352; for dermal contact: Cr = 6.00 × 10−5, Cu = 0.012, Zn = 0.06, As = 1.23 × 10−4 and Pb = 0.000525 [Ingestion of particles   are the DDermal) ,37. Thus0.000525 ,39,40,41The non-carcinogenic effect for n number of heavy metals, on the population, is the sum of all HQs, represented as the Hazard Index (HI), . Hence, ing + LAADinh + LAADderm) is the lifetime average daily dose expressed as a weighted average for each exposure path, SF is the slope factor for a particular carcinogenic element [−4 to 1.0 × 10−6 is considered an acceptable level [−4 then, it is considered a lifetime carcinogenic risk to the person exposed. Detailed indices (non-carcinogenic and carcinogenic) are computed in −6 ; 1 × 10−6 to 1 × 10−5 ; 1 × 10−5 to 5 × 10−5 ; 5 × 10−5 to 1 × 10−4 ; 1 × 10−4 to 5 × 10−4 ; 5 × 10−4 to 1 × 10−3 ; >1 × 10−3 .On the other hand, carcinogenic risks (CR) are estimated by the Equation (8): element ,40,43. Sle level  but whenEstimated Daily Intake (EDI) of metals, Target Hazard Quotient (THQ), Hazard Index (HI), Cancer Risk (CR) and Total Cancer risk (TCR) are the indices addressed to estimate probabilistic risk due to consumption of vegetables grown in contaminated soil and detailed of the indices are computed in The concentration of heavy metals  in the industrially affected soil along with control soil is presented in geo), contamination factor (CF) and Pollution load index (PLI), and measured values are computed in Environmental ecological risk by the HMs  was assessed by calculating single indices such as, enrichment factor (EF), geo-accumulation index  of the studied HMs are summarized in The pollution load index (PLI) was calculated to assess the integrated index of pollution by heavy metals in the contaminated soil, which is depicted in Cluster analysis is designed for the better identification of a distinguishable group of items at the sampling site against the detected parameters with respect to notable variability . An almoThe principal component analysis (PCA) was conducted to determine the correlation and retrospective sources of the tested elements ,25. The The concentration of HMs  present i−n the examined vegetable samples are illustrated in The high level of Pb and As in the plant species may be explained by the pollutants present in irrigation water, land texture, used fertilizer or due to pollutants from highway traffic and the industrial establishment around the sampling site . HoweverThe overall heavy metal pollution in the various studied vegetables is estimated by calculating MPI . The higThe transfer of HMs from soil to plant (BCFs) depends on the soil physicochemical characteristics; types of HM accumulation and plant species . Heavy mThe adverse effect of HMs contaminated soil on human health (carcinogenic and non-carcinogenic) through ingestion, inhalation and dermal contact and health risk (carcinogenic and non-carcinogenic) due to consumption of HMs contaminated vegetables for both adults and children are calculated and computed in −2; child: 6.15 × 10−1) and lowest for Zn . In contrast, for the path inhalation, maximum HQ value was found for Cr  and minimum for Zn , while for dermal contact maximum HQ value was found for As  and minimum for Zn , respectively. However, the possible non-carcinogenic risk effect of HMs contaminated soil exposure (HQsoil) through all three paths can be ranked in the order of As > Fe > Cr > Pb > Cu > Zn for adults, with a similar trend for the child as well, but in each case, the estimated value was found higher in children compared to adult  is found higher in children than adults in each case, which is consistent with the finding by Proshad et al. [−4 and 1.81 × 10−5 for As and Pb, respectively, for the child, while those for adults were 4.16 × 10−4 and 4.50 × 10−6, respectively, in the agricultural soil of Jhinaidhah and Kushtia district of Bangladesh.In this study, health risks due to direct soil exposure are calculated considering average metal concentrations  of affected soil in the ten sampling sites and computed in to adult , which cnd child  indicatind child  for heavble risk ,45. Furty action ,45. On tle range ,45. In ar et al.  for chilr et al.  estimated et al. , where t−4) > Cu(1.24 × 10−5) > Zn(9.80 × 10−6) > Pb(3.42 × 10−6) > As(1.70 × 10−6) > Cr(6.70 × 10−7) and Fe(2.39 × 10−4)> As(2.70 × 10−5) > Cu(1.95 × 10−5)> Zn(1.40 × 10−5) > Pb(5.00 × 10−6) > Cr(1.00 × 10−6) for adult and child , indicating no potential non-cancer risk from the vegetables upon consumption by both the population group. However, it is mention-worthy that, in each case, the THQ values for children are higher than the adult. This scenario is also consistent with the findings of [−9, 2.55 × 10−8, 2.92 × 10−8 for adult and 3.16 × 10−9, 4.02 × 10−8, 4.58 × 10−8 for child, respectively. All the CR values are below the threshold limit of > 1 × 10−6 and according to Li et al. [In general, a variety of vegetables are consumed by different population segments throughout the year. Thus, estimation of the average intake of metal from the different varieties of vegetables is more realistic, therefore, the mean concentration of metals  in the 15 varieties of vegetables are considered for the calculation of health risk indices  in this study, which are computed in nd child , respectdings of . In a prdings of  reportedi et al. , CR valui et al.  and Bour5) > Zn9. × 10−6 >−1 and 1.09 for adults and children, respectively. Lemly [−3 and 4.05 × 10−3 for adults and children, respectively, appraising HI < 1 revealing no risk. Similarly, total lifetime carcinogenic risk value (TCR) for soil and vegetable for both the population groups were measured , Radish (0.5435), Coriander (0.5157), Brinjal (0.4711) and Cauliflower (0.3156) for Pb, indicating metal contamination by anthropogenic activities and suggested regular monitoring. Estimated HQ via direct soil exposure can be ranked as HQing > HQderm > HQinhel regardless of age, and HQ values for all the elements in the entire three pathways for adults and children were <1, indicating not to pose any health effect. Similarly, HQ via vegetable consumption was found below unity for both the population group and recommended safety limit. Nonetheless, HI value via direct soil exposure was measured <1 for adults and >1 for the child, on the other hand, total lifetime carcinogenic health risk for adults lied within level II (1 × 10−5 to 1 × 10−6) but in Level-V (1 × 10−4 to 5 × 10−4) for children, stipulated medium to high risk. In contrast, HI and TCR values for the population group via dietary intake of vegetables collected from the industrially affected soil site found within the safety limit recommended by international bodies.This study has assessed heavy metal contamination in the soil of agricultural land adjacent to an industrial zone of Ashulia, Savar, Bangladesh and their accumulation in the cultivated vegetables on that field. The elevated level of HMs  were found in the industrially affected soil compared to control soil and the estimated value of EF, I


Lilium pumilum is an important ornamental, culinary and medicinal bulbous plants with salt tolerance. However, salt tolerance of lily, particularly the bulb, has been studied relatively little, which brings challenges to the cultivation of lily varieties with high salt tolerance. Here, we performed transcriptome sequencing on the bulb organs of L. pumilum under salt stress treatment, analyzed differential gene expressed levels and then identified several key genes associated with salt stress tolerance at genome-wide scale. For the first time, we revealed the obvious response against salt stress for L. pumilum bulb organs, while distinct from those for root organs. Several key genes obtained through transcriptome analysis and DEG screening include NF-YB3 transcription factor, metallothionein type 2 protein, vicilin like seed storage protein and bidirectional sugar transporter SWEET14. Rather than typical ROS scavengers like superoxide dismutase, peroxidase, and glutathione transferase, non-typical ROS scavengers such as the metallothionein type 2 protein, and vicilin like seed storage protein were upregulated in our work. The bidirectional sugar transporter SWEET14 protein and the hormone signaling proteins such as E3-ubiquitin protein ligases, PYL4 and protein phosphatase 2C were also upregulated, suggesting the role of sugars and hormones in the bulb organ responses to salt stress. Co-expression analysis of the DEGs further confirmed that NF-YB3 transcription factor acted as a hub gene, suggesting that salt stress can promote flowering of L. pumilum. Taken together, we identified important candidate genes associated with salt tolerance of the L. pumilum bulb organs, which may provide the excellent basis for further in-depth salt tolerance mechanisms of the lily bulbs. Lilium pumilum, commonly known as coral lily, is native to grassy mountainous areas of northeast China which are heavily salinized region of the country. Therefore, L. pumilum is regarded as a salt tolerant plant, but the molecular mechanism of salt tolerance in L. pumilum has not been extensively studied, which limits its widespread application.Lilium, a genus of herbaceous flowering plant growing from bulbs, is widely cultivated horticultural crop. The flowers of these plants are popular in ornamental gardens or indoor potted plants. Lilies are also used in the culinary industry and folkloric medicine . Lilium + exclusion and vacuole compartmentalization are another typical plant responses to minimize the ion toxicity , calcineurin B-like proteins (CBLs) with CBL-interacting protein kinases (CIPKs) and calmodulin-binding transcription activators (CAMTAs), transcription factors such as basic leucine zipper (bZIP), WRKY, APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF), MYB, basic helix–loop–helix (bHLH) and NAC families, Na+ and K+ transporters like CYCLIC NUCLEOTIDE-GATED CHANNEL (CNGC) and the GLUTAMATE-LIKE RECEPTOR (GLR) families, tonoplast-localized Na+/H+ exchanger 1 (NHX1), plasma membrane-localized SALT OVERLY SENSITIVE 1 (SOS1) Na+/H+ antiporters, high-affinity K+ transporter HKT, ROS scavenging enzymes like superoxide dismutase, peroxidase, and glutathione transferase, organic osmolytes like proline, glycine betaine, sugar alcohols, polyamines, and proteins from the late embryogenesis abundant (LEA) superfamily  can provide dynamic nature of transcriptome at higher coverage and greater resolution than previously employed methods such as Sanger sequencing- and microarray  in the L. pumilum bulb organs upon salt stress treatment were then identified by comparing the gene transcript profiles in the bulb organ samples between salt-treated and control plants at different time points of salt stress treatment, mainly focusing on identifying the candidate genes associated with transcription regulation, ROS scavenging, ion detoxification, osmolyte accumulation and hormone signaling. Putative hub genes were predicted through building co-expression network using STRING database and Cytoscape software. The expression levels of several key genes were validated by qRT-PCR. The preliminary results of this study provided the basic information on molecular mechanism underlying regulatory network for salt stress responses in the L. pumilum bulb organs that may serve as foundation for further in-depth functional studies.In this study, we analyzed the transcriptome data of the L. pumilum plants were grown in laboratory at College of Landscape Architecture of Northeast Forestry University . Healthy seedlings were cultured on hydroponic tank supplemented with Hoagland nutrient solution at 70% relative humidity, and 22-25 °C ambient temperature. 3 wk-old plants were treated with 150 mM NaCl for 12 h. The plants at 0 h of NaCl treatment were used as control. Bulb organ samples were collected at 2 h and 12 h, respectively.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trinity 2.4.0 , Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Pfam, swiss-Prot, and eggNOG database using DIAMOND . The quantity and purity of the extracted RNA samples were evaluated by Bioanalyzer 2100 and RNA 100 Nano LabChip Kit  with RIN number >7.0. PolyA RNA was obtained by purification of total RNA with oligo-dT magnetic beads. Then, mRNA was treated with divalent cations under temperature gradient for fragmentation into smaller size. Next, cDNA library was constructed by reverse-transcription reactions from those cleaved RNA fragments using RNA-seq sample preparation kit . Finally, the cDNA library was sequenced on Illumina Novaseq™ 6000 platform . All raw reads were deposited in the Sequence Read Archive (SRA) database in NCBI with accession number PRJNA851552. Clean reads were obtained by removing adaptor contamination, low quality bases as well as undetermined bases using Cutadapt  and in-hty 2.4.0  was util DIAMOND  with thrL.pumilum.Expression levels of the unigenes were determined by calculating TPM (Transcripts Per Kilobase of exon model per Million mapped reads) . DEGs wehttps://cn.string-db.org/cgi/input?sessionId=bOrBIG9AvMjL&input_page_show_search=on ). The Arabidopsis thaliana was used as reference organism in the prediction. Then, the network was built using Cytoscape. Network analysis and hub gene identification were conducted using network analyzer and cytohubba plugins, respectively, in Cytoscape.In order to build co-expression network. The DEGs at 0-12 h with p <0.05 were selected and their interaction was predicted using STRING database  and were synthesized by Comate Bioscience . Primer information was shown in Additional file. qRT-PCR was performed using triazole reagent UltraSYBR Mixture kit . The 20 μl reaction system was as follows  by a reaction flow of 37 °C, 15 min and 98 °C, 5 min. Primers were designed using the RealTimePCR design tool of the IDT website  according to following program: preincubation, 95 °C for 10 min; three steps amplification, 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 32 s ; melting, 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s; cooling, 37 °C for 30 s. Lily L. pumilum were sequenced by Illumina double-terminal sequencing technology. Three biological replicates, a total of 9 samples, were generated for each time point. The sequencing data of all samples exceeded 6 G. In total, 492572310 raw sequencing reads were obtained from the control and treated samples and were then subjected to cleaning overrepresented sequences such as Illumina adaptor sequence, generating 455124918 clean reads that were used for subsequent analyses  and Phoenix dactylifera (19.58%)  were annotated to NR. According to the annotation result to NR database, the unigenes of (19.58%) Figure 2L. pumilum. In the categories of “cellular component” and “molecular function”, various cellular components and molecular functions such as nucleus, cytoplasm, membranes, chloroplast, and mitochondria and protein binding, DNA binding, metal ion binding, etc were enriched in salt stress response of L. pumilum. In the KEGG annotation, gene transcription and translation also showed the highest activities and environmental adaptation and secondary metabolilsm were the most active metabolism except for the housekeeping metabolisms such as carbohydrate, lipid, amino acid metabolisms  were identified by pairwise comparison of TPM values. 1772 (878: upregulated and 894: downregulated) DEGs were identified at 0-2 h period of salt stress, and 2324 (1031 upregulated and 1293 downregulated) DEGs at 2-12 h period of salt stress , total 4096 DEGs  of salt stress by clustering the DEGs according to the similarity in their functions, metabolic processes or cellular pathways. In our study, TPM of a differential gene was displayed as Z-value, and the cluster analysis was performed using 100 DEGs with the smallest analysis Figure 7The DEGs at the initial stage (0-2 h period) and later stage (2-12 h period) during salt stress were subjected to GO enrichment analysis Figure 8DEGs were highly enriched in the GO functions such as “biological process”, “translation”, “DNA-templated transcription and its regulation”, “oxidation-reduction processes”, “nucleus”, “cytoplasm”, “membrane”, “protein binding”, “DNA binding”, etc. The different enrichment results at the initial stage (0-2 h) and the later stage (2-12 h) indicated that different genetic programs controlled the response of lily bulb to salt stress in this study. As the salt stress prolongs, the genes that belong to the GO functions including “cytoplasmic translation”, “response to heat”, “proteosome-ubiquitination dependent protein catabolism”, “proteolysis involved in cellular protein catabolic process”, “response to auxin”, “response to oxidative stress”, “protein folding”, “response to cadmium ion”, “transmembrane transport”, “cell wall organization”, “cell division”, “chromatin silencing”, “flavonoid biosynthetic process”, “flavonoid glucuronidation”, “cytosolic small ribosomal subunit”, “plasmodesma”, “cellular component”, “intracellular”, “apoplast”, “plant type-cell wall”, “zinc ion binding”, and “protein serine/threonine kinase activity” showed dramatically differential expression. Therefore, it is obvious that the complicated responses elicited by salt stress includes a wide-range of cellular activities and the genetic program of the plant (lily bulb) has a temporal differential pattern in which the aforementioned DEGs are regulated differently depending on time.In addition, KEGG pathway annotation of the DEGs in two different periods (0-2 h vs 2-12 h) during salt stress showed that the DEGs were associated with more than 30 pathways and were highly enriched in the pathways associated with “Ribosomes”, “Plant hormone signal transduction”, “Phenylpropanoid biosynthesis”, “MAPK signaling pathway-plant”, and “Starch and sucrose metabolism” Figure 9Then, we focused on the 958 DEGs which were co-induced throughout the entire period of salt stress (0-12 h) , and only 25 genes with annotated information showed temporal differential expression , hormone signaling , primary metabolism for energy conservation (sulfite reductase flavoprotein), secondary metabolite production , seed development (vicilin like seed storage protein), and transcription factors . In the whole process of salt stress, the expression of bHLH84, bHLH123, bHLH123*, bZIP19, homeobox-leucine zipper protein ROC3, HSFC1 was downregulated, and the expression of NF-YB3 was upregulated 3-6 times. Among other DEGs, metallothionein type 2 protein showed unusually high expression levels and strong upregulation in response to salt stress treatment.Total 1039 DEGs at 0- 12 h (p< 0.05) were chosen as input data for prediction of their interaction in STRING database. Using Cytoscape, we input the output file of STRING search and obtained a network with 432 DEGs and their 1126 interacting events. We further ranked them and identified putative hub genes using cytohubba plugin , MED37C (TRINITY_DN32748_c0_g1), PER1 (TRINITY_N30585_c1_g4), ADH1 (TRINITY_DN34595_c0_g1), ZAT6 (TRINITY_DN29763_c0_g1), BT2 (TRINITY_DN28127_c0_g3), KING1 (TRINITY_DN32088_c0_g2), NF-YB3 (TRINITY_DN19275_c0_g1), HSP70-18 (TRINITY_DN21721_c1_g4), TPL (TRINITY_DN30465_c0_g4). Among them, TPL and NF-YB3 showed relatively higher log2FC values  in our RNA-seq data, suggesting that they play inductive and key roles in the response of lily bulbs to salt stress.To verify accuracy and reliability of the RNA-seq data, we randomly selected twelve genes and further quantified their expression levels by qRT-PCR. The results showed that the expression patterns of the DEGs determined through qRT-PCR were highly consistent with the RNA-seq data  were obtained by Illumina sequencing of total RNA samples, and analyzed. A total of 51566 unigenes were found and 1772 and 2324 DEGs were identified at initial (0-2 h) and later (2-12 h) periods, respectively. GO and KEGG enrichment analysis for these DEGs indicated that expression patterns of DEGs and KEGG annotated pathways showed slight difference between 0-2 h and 2-12 h. Among the DEGs, 958 DEGs were co-expressed during entire period (0-12 h) of salt stress, and their expression patterns were analyzed. It was found that many unigenes annotated as transcription factors were involved in the responses to salt stress. They belong to AP2/ERF, bHLH, bZIP, HD2/ZIP2, HSF, MYB, NAC, NF-YB and WRKY families. Among them, NF-YB3 transcription factor showed the most intense upregulation throughout entire period of salt stress  and osmolyte accumulation-related proteins . Besides, phytohormone signaling factors (E3-ubiquitin protein ligases and protein phosphatase 2C) were upregulated, suggesting that salt tolerance of the lily plants requires hormone signaling. Among the DEG transcription factors including DREB, ERF, bHLH, bZIP, HD2/ZIP2, HSF, MYB, NAC, NF-YB, and WRKY  can be found below: KS, UP, and SS conceived and designed the experiments. KS and SS conducted the experiments. KS, UP, YW, and HY elaborated and analyzed experimental data, YZ, KS, and UP wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Fundamental Research Funds for the Central Universities [grant numbers 2572019BK04]; and the Natural Science Foundation of Heilongjiang Province [grant numbers LH2019C004].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In addition to reducing the concentration of nitrate (77.21%), NaCl salt stress increased the concentrations of key bioactive molecules, notably carotenoids (lutein and β-carotene), phenolic acids, and flavonoid derivatives, thus resulting in a higher antioxidant activity of salt-treated basil plants compared to the untreated ones. Analysis by UHPLC revealed that cichoric acid was the most abundant polyphenolic compound in all basil cultivars, with the highest values recorded in ‘Cinnamon’.Depending on duration and magnitude, abiotic stresses interfere with plant metabolic processes and may severely impact developmental and qualitative attributes. In this study, in addition to characterizing three different cultivars of basil  grown under hydroponics, we appraised the impact of NaCl salt stress (60 mM) on morphophysiological and nutraceutical properties of the basil crop. Salt stress significantly reduced fresh yield  and photosynthetic parameters (ACO The world population has peaked at eight billion people, and recent estimates project an increase of more than two billion people by 2050. In the coming decades, the imperative for community policies will be to ensure food security, a daunting challenge when we consider that it is mainly dependent on agricultural production . CurrentSalvia mirzayanii and Salvia acrosiphon and by Perin et al. , mineral profile (by ion chromatography) and phenolic profile (by ultrahigh performance liquid chromatography).Currently, in developing countries, about 80% of medicines are of plant origin, while in developed countries, the proportion is only 25%. Among medicinal plants, basil (he years . Its famhe years . Not surOcimum basilicum L.), was carried out in spring–summer 2021 in the greenhouses of the Federico II University of Naples Department of Agriculture (DIA) . On 5 May 2021, basil plants ‘Anise’ , ‘Cinnamon’  and ‘Lemon’  were seeded in 54-hole polystyrene trays  until two true leaves appeared. At 14 days after sowing , seedlings were transplanted into round anti-spiraling pots (0.1 m × 0.1 m × 0.15 m) filled with a mixture (v/v) of 1/3 perlite and 2/3 peat  (−2. Nutrient solution (NS) was distributed through drippers with a flow rate of 2 L h−1 (1 dripper/plant).The experimental trial, aimed at evaluating the effects of NaCl salt stress in basil  . The pot3-N, 1.0 mM NH4-N, 1.5 mM P, 5.0 mM K, 1.75 mM S, 4.5 mM Ca, 2 mM Mg, 9 μM Mn, 20 μM Fe, 0.3 μM Cu, 20 μM B, 1.6 μM Zn, and 0.3 μM Mo. Each experimental unit was replicated three times and included 15 plants (45 plants per treatment). Saline NS was prepared by adding 60 mM NaCl to the control NS.Basil seedlings were arranged in the greenhouse according to a bifactorial design in which three basil cultivars  and two NSs  were considered as factors. The control NS was a modified Hoagland with the following nutrient element composition: 13.0 mM NO−1). The height (cm) and number of leaves per plant were then determined. The collected samples were dried in a ventilated oven at 60 °C until constant weight (about three days) for the measurement of the dry weight (g plant−1) and the percentage of dry matter. The dried plant material was then finely ground using an MF10.1 cutting head mill  for the measurement of the mineral concentration. Four plants per replicate were sampled and immersed in liquid nitrogen, stored at −80 °C, and subjected to a freeze-drying cycle  for the measurement of antioxidant activity, carotenoids, and phenolic acids, while another part was stored at −20 °C for the measurement of chlorophyll concentration.At harvest (26 days), eight plants per replicate were cut at root collar and sampled to determine biometric parameters and yield. Freshly sampled plants were weighed for total fresh and leaf weight measurements  for distortion correction and brightness and contrast adjustment. Leaf area was quantified using ImageJ v1.52a software . The analyses were performed in triplicate and the leaf area was expressed in cm2.Digital quantification of the leaf area was performed using photos of the leaves of each plant sampled. Specifically, at 26 days, the leaves of each plant were placed on a white panel perpendicular to the camera lens . The captured photos were processed with Adobea*, b* (CIELAB) color space, where L indicates brightness, a and b chromaticity . Chroma and Hue angle (°) were calculated according to the formulas proposed by Kheng [Just before harvest, colorimetric indices were determined on ten healthy and fully expanded leaves per replicate using a Minolta CR-400 portable colorimeter . Color was converted according to the CIE 1976 L, by Kheng .(1)ChroAt 26 days, six fully expanded and well illuminated leaves per replicate were labeled and used to determine gas exchange and leaf fluorescence. Measurements were taken in the middle of the day (11:00 a.m.–3:00 p.m. solar time).2 assimilation rate , transpiration , and stomatal conductance  were measured with the LI-6400 portable gas exchange system . The CO2 in the measurement chamber was set at ambient values (about 400 ppm) and the photosynthetically active radiation at 1000 µmol m−2 s−1. The leaves were closed in the measurement chamber until equilibrium was reached (about 15 min).The net COv/Fm Meter fluorometer  on dark-adapted leaves for 10 min using leaf clips. Maximum chlorophyll fluorescence (Fm) was induced with a saturating light pulse of 3000 μmol photons m−2 s−1 for 1s, while initial fluorescence (Fo) was recorded with an internal blue LED light of 1–2 μmol photons m−2 s−1. Fv/Fm was estimated as (Fm − Fo)/Fm.Chlorophyll fluorescence measurement was performed using a portable Fβ-carotene) concentrations were determined by spectrophotometry and high-performance liquid chromatography with diode array detection (HPLC-DAD), respectively. All reagents were purchased from Sigma-Aldrich . Analyses were performed in triplicate.The concentrations of leaf chlorophyll and carotenoid (lutein and v/v) and homogenized by IKA® T10 basic Ultra Turrax® homogenizer . Chlorophyll a and b concentrations were determined using a UV-Vis spectrophotometer  with an absorbance of 647 and 664 nm, respectively. Total chlorophyll was calculated as chlorophyll a + chlorophyll b and expressed as mg g−1 of fresh weight (fw).To determine chlorophyll according to Wellburn ’s protocβ-carotene concentrations were determined in freeze-dried basil leaves according to the protocol of Salomon et al. [v/v) and subjected to a vacuum centrifugation cycle to separate the pellet from the solvent. The pellet was mixed with methanol and MTBE (methyl-t-butyl ether) in a 1:1 (v/v) ratio and analyzed by HPLC-DAD. The results were expressed as mg kg−1 of dry weight (dw).Lutein and n et al. . A 0.1 gTo determine the antioxidant activity, three spectrophotometric methods were compared: DPPH , ABTS ), and FRAP (ferric ion reducing antioxidant power), according to protocols of Brand-Williams et al. , Re et aTo determine the antioxidant activity of DPPH, 1 mL of DPPH solution (0.1 mM) and 96% methanol were added to 200 μL of the aqueous extract, mixed and incubated at room temperature for 30 min in the dark. The absorbance was recorded against the blank at 517 nm.+ was prepared by reacting 7 mM aqueous solution of ABTS+ with 2.45 mM aqueous solution of potassium persulfate in equal parts. After incubation in the dark (16 h at 23 °C), the stock solution was diluted with ethanol in a ratio of 1:88 until an absorbance of 0.700 ± 0.020 at 734 nm was reached. A 0.1 mL aliquot of each sample, previously filtered and diluted (1:10) with 70% methanol, was mixed with 1 mL of ABTS+ solution and stored at room temperature for 2.5 min. The absorbance was read at 734 nm.To determine ABTS antioxidant activity, a stock solution of ABTS2+/2,4,6-tris (2-pyridyl)-s-triazine (10 mM) in HCl (40 mM) + 1.25 mL of FeCl3 (20 mM) in water + 12.5 mL of acetate buffer . An aliquot of 150 μL of the sample was mixed with 2.850 mL of FRAP solution and incubated for 4 min in the dark. The absorbance was read at 593 nm against a blank containing all the reagents.For measurement of FRAP antioxidant activity, a FRAP solution was prepared containing 1.25 mL of Fe−1 dw. All analyses were performed in triplicate.The absorbances of the DPPH, ABTS, and FRAP essays were recorded with a UV-vis spectrophotometer . The results were expressed as Trolox equivalent mmol kgTM, Waltham, MA, USA) and Orbitrap high-resolution mass spectrometry  according to the protocol detailed by El-Nakhel et al. [v/v). The Q-Exactive Orbitrap mass spectrometer was set in a fast negative/positive ion switching mode with two scan events  for all compounds of interest. The calibration and accuracy of the equipment was monitored using a standard reference mixture . Data processing was performed with Quan/Qual Browser Xcalibur software, v. 3.1.66.10 . Polyphenols were expressed as mg kg−1 dw.For the measurement of the phenolic profile, 5 μL samples extracted according to the procedure described by Vallverdu-Queralt et al.  were anal et al. . The chrTM DionexTM, Sunnyvale, CA, USA) according to the detailed protocol of Formisano et al. [TM 6.8 Chromatography Data System software  data of samples with those of certified reference standards.The measurement of cations  and anions  of basil leaves was carried out by ion chromatography coupled with an electrical conductivity detector  for Microsoft Windows 11. A two-way analysis of variance (ANOVA) was applied to assess the significance of the effects and interactions between the cultivar factors (CV) and salt stress (S). The mean effect of CV factor was compared by one-way analysis of variance, and the mean effect of S was compared by Student’s −1). Compared to the Control, Salt treatment reduced the number of leaves by 39.15, 16.11, and 44.54% in ‘Anise’, ‘Cinnamon’, and ‘Lemon’, respectively  and Hue angle (+2.21%) was observed in plants under salt stress, compared to the Control, while b* (−26.49%) and Chroma (23.79%) decreased. In ‘Anise’ and ‘Cinnamon’, Salt treatment did not result in significant differences for a* and Chroma. An opposite trend was observed for the hue angle between the Anise and Cinnamon cultivars. Specifically, compared with Control, the above colorimetric parameter was not affected by salt treatment in ‘Anise’, while in ‘Cinnamon’ there was a 1.90% increase.As shown in p < 0.001) reduced net CO2 assimilation (ACO2), transpiration (E), and stomatal conductance (gs) by 23.72, 30.95, and 29.16%, respectively. The same parameters were influenced by the mean effect of the cultivar. Specifically, ‘Lemon’ showed the highest E and gs values compared to ‘Anise’, but the lowest ACO2 values compared to ‘Cinnamon’. On the contrary, the maximum quantum efficiency of photosystem II (Fv/Fm) was solely affected by S. The data in v/Fm values (3.79%) compared to the control.The CV × S interaction did not result in significant differences for the physiological parameters shown in β-carotene . Compared to the control, salt stress increased total phenolics by 21.63%.As shown in Like total phenols, phenolic acid derivatives and total flavonoids showed a similar trend in response to salt stress B,C. Rela−1 dw; Compared to total flavonoid derivatives, rutin was not significantly affected by the interaction of the factors considered (CV × S), unlike what was observed for di-hydroquercetin glucoside and quercitin glucoside. Regarding these glycoside compounds, salt stress increased their concentrations in ‘Anise’ and ‘Cinnamon’, while no significant differences were observed in ‘Lemon’. In contrast, in ‘Lemon’, salt stress increased the concentration of quercetin glucoside . Specifically, salt stress, compared to the control, increased the concentrations of the above acids by 16.73, 21.28, and 31.22%, respectively. Relative to the mean effect of the cultivar, ‘Anise’ recorded the highest concentrations of cichoric and rosmarinic acids, while ‘Cinnamon’ had the highest content of feruloyl tartaric acid .Similarly to the findings for the above phenolic acids, the CV × S interaction was not significant for salvianolic K, salvianolic L, and chlorogenic acids. However, salt stress significantly increased the concentrations for the acids mentioned above compared to the control. Regardless of stress, ‘Anise’ was characterized by the highest concentration of salvianolic L and chlorogenic acids, contrary to what was observed for salvianolic K acid, which did not show a significant difference between cultivars .Unlike what was observed for the cichoric, rosmarinic, feruloyl tartaric, salvianolic K, caffeic, and salvianolic L acids, the concentrations of caftaric, salviananic acid A, chlorogenic, and salvianolic A acids were significantly affected by the CV × S interaction.−1 dw). The same interaction also resulted in the highest concentration of caftaric (81.68 mg kg−1 dw) and caffeic acids (140.57 mg kg−1 dw). In ‘Anise’ and ‘Cinnamon’, compared with the control, salt stress increased salvianic acid A concentrations by 32.36 and 15.50%, in contrast to ‘Lemon’, which did not show a significant difference.Salt stress increased salvianolinic acid A concentration in all cultivars, with the highest values obtained by Anise × Salt (16.68 mg kg−1 dw) while ‘Anise’ had an 8.7% higher K concentration than ‘Cinnamon’ (The effects of the factors considered (CV and S) and their interaction (CV × S) on the accumulation of minerals in basil leaves are shown in innamon’ . Regardlinnamon’ , while ainnamon’ . Compareinnamon’ . The saminnamon’ .Salvia officinalis L. [Mentha spicata L. [2 assimilation rate and transpiration. Like Bekhradi et al. [v/Fm values that never reached critical values , ‘Cinnamon’ was the most productive cultivar, followed by ‘Anise’ and ‘Lemon’ . Howevernalis L.  and Menticata L. . Regardlicata L.  consideri et al.  and Attii et al. , salt-inl values D. Howevenditions , it may The analysis of morphological parameters of the salt-treated plants showed a lower sensitiveness of the Anise and Cinnamon cultivars than ‘Lemon’. The latter reduced leaf area and height by 57.38 and 35.37%, compared to the control; in contrast, ‘Anise’ and ‘Cinnamon’ reduced, on average, the same parameters by 33.28 and 19.87%. The above is confirmed by the reduction in total dry weight in ‘Lemon’ (56.97%) in the salt treatment compared to the control. In contrast, no significant changes were observed in ‘Cinnamon’ for this key parameter. This result could be partially attributable to a constitutive higher tolerance to salt stress and partially to the increase in total dry matter, which increased from 9.27% (Cinnamon × Control) to 15.10%  E. As Ber+ and Cl− in transpiration fluxes, leading to a reduction in total fresh weight  would suggest that Na+ accumulation was not totally internalized in the leaves, a condition that, as reported by Attia et al. [+ concentrations reduce K, Mg, and Ca uptake, only a reduction in K was observed in our experiment under salt stress; differentially, the concentration of Mg and Ca increased. As Scagel et al. [+, the use of 60 mM NaCl in the nutrient solution increased the Cl concentration in the leaves, reaching an average value of 36 g kg−1 dw in the ‘Anise’ and ‘Cinnamon’, which explaining the observed worsening production performance , an antinutritional compound with a negative impact on human health. In salt treatment, the acknowledged antagonism between Cl− and nitrate would have reduced the latter’s uptake, slowing the plants’ growth rate [Although Attia et al.  showed tcultivar . This rea et al. , would el et al.  suggesteformance . In factectively . As obsewth rate . Althougwth rate , regardlwth rate , this rewth rate . HoweverUnlike in ‘Lemon’, salt significantly altered colorimetric parameters  comparedβ-carotene) than ‘Lemon’, it had lower mean values of DPPH, ABTS, and FRAP. This result could be attributable to the higher constitutive concentration of flavonoids in ‘Lemon’, which by structural characteristics may have positively influenced the antioxidant activities of DPPH, ABTS, and FRAP.‘Lemon’ was characterized by a 160.57% higher total concentration of flavonoid derivatives compared with ‘Cinnamon’. This remarkable difference partially justifies the significant colorimetric differences observed between cultivars, as flavonoids also confer color to vegetables ; what haDespite the phylogenetic distance and significant differences in yield and quality, the three basil types  tested had similar responses to NaCl salinization. Specifically, salinized plants enacted adaptive stress responses mainly based on reduced photosynthetic activity and growth  but at the same time increased secondary metabolites, increasing their antioxidant properties. Specifically, as shown in