Temozolomide combined with whole-brain radiotherapy has good near-term efficacy and safety in the treatment of brain metastases from nonsmall cell lung cancer. In this study, we analyzed the risk factors for treatment and prognosis of brain metastases in gestational trophoblastic neoplasm (GTN) during pregnancy. Thirty-one patients with brain metastases were included in the study. All patients had a pathological diagnosis of primary lesions, including 23 adenocarcinomas, 7 squamous carcinomas, and 1 adenosquamous carcinoma, and had ≥3 intracranial metastases, controlled primary lesions /complete remission (CR) for ≥2 months) by cranial enhancement MRI and no extracranial metastases. Presence or control of extracranial metastases was for ≥2 months. The common adverse toxic effects were nausea, vomiting, neutropenia, and thrombocytopenia, but most patients tolerated them with symptomatic management. Gestational trophoblastic neoplasm (GTN) is the only gynecologic malignancy associated with pregnancy that can be cured by chemotherapy. To date, the incidence of GTN has not been clearly documented and varies widely between different regions of the world –4, with Basic research  and targGTN is as immunogenic as normal gestational trophoblast cells and stimulates an immune response from the maternal immune system, but GTN successfully evades immune surveillance and modulates the maternal placental and peripheral immune systems and then rapidly proliferates, grows, invades, and metastasises widely. Drug resistance or refractory to treatment is the main reason for the failure of GTN treatment, and therefore, immunotherapy is becoming a hot topic for exploratory research in this area .n = 63), epithelial GTN tissues (n = 12), and placental GTN tissues (n = 41) were found to be positive for PD-L1, most of which were strongly positive [By analyzing the expression of PD-L1 in the tumor tissues of GTN patients, all choriocarcinoma tissues  GTN treated with pabolizumab was reported to have a favourable outcome . The patThe patient was treated with pabolizumab 200 mg (21 d in 1 cycle). At the time of writing, 3 courses of treatment had been administered and the symptoms associated with brain metastases had resolved significantly, and HCG had decreased to normal levels. After 2 courses of pabolizumab treatment, hepatic impairment of degree III was observed, but liver function returned to normal with hepatoprotective therapy and did not affect subsequent treatment. No other grade III-IV drug toxicities were observed during pabolizumab treatment. Four patients with GTN at very high risk of liver or brain metastases were reported to be treated with pabolizumab. All patients were treated with pabolizumab 2 mg/kg for 21 d for 5 courses, resulting in complete remission in 3 patients and death due to progression in 1 patient . Two casWith the advancement of molecular typing of malignant tumours and hereditary tumours and the rapid development of biologically targeted therapies, the treatment of malignant tumours has gradually entered the era of precision medicine. In recent years, the clinical application of targeted therapies in GTN has also made breakthroughs.The expression of VEGF in GTN tissues was found to be significantly higher in normal placental tissues than in staphyloma tissues, and the expression of VEGF, angiopoietin-1, and angiopoietin-2 was also significantly higher in placental GTN tissues than in choriocarcinoma tissues . AntiangThe epithelial growth factor receptor (EGFR) is a transmembrane glycoprotein located on the surface of the cell membrane and is a member of the ErbB family of conserved receptors. EGFR acts by influencing tumour cell proliferation, invasion, metastasis, and apoptosis. In vitro studies found that EGFR and HER2 were significantly overexpressed in GTN cells and that lapatinib significantly inhibited tumour cell growth by regulating the expression of EGFR and HER2 in GTN. To investigate the expression pattern of c-ErbB2 and Bcl-2 proteins in staphylococcal tissues, they performed immunohistochemical analysis on 220 pregnancies, including 39 aborted tissues and 41 partial and 140 complete staphylococcal tissues [Current research in GTN targeting also includes targeting abnormal pathways, such as the PI3K/AKT pathway. Activation of the PI3K/AKT signaling pathway was found to stimulate the proliferation of GTN cells, while lapatinib inhibited the proliferation of GTN cells by blocking the PI3K/AKT signaling pathway . The mecThe incidence of brain metastases was 0.36% in 3 cases and renal metastases was 0.12% in 1 case. The incidence of metastases to the brain was 0.95% in 735 patients with choriocarcinoma and 1.1% in 8 patients with metastases to the kidneys; 2 patients had metastases to both the brain and kidneys, with an incidence of 0.27%. In this group of 19 patients, the age ranged from 22 to 48 years old, with an average of 29.8 years old.In 5 cases, the last pregnancy was gravida, in 10 cases, early pregnancy, in 2 cases, full-term delivery, in 1 case, ectopic pregnancy, and in 1 case, rupture of the right uterine horn. 17 of the 19 cases had pulmonary metastases, 8 cases had cerebral metastases, 2 cases had vaginal metastases, and 4 cases had pelvic organ metastases.All patients underwent blood tests for HCG and 13-IaCC, abdominal ultrasound or CT, pelvic ultrasound, lung CT, or lung X-ray. Some patients underwent cerebroscopy and intravenous pyelogram.Four patients were critically ill at the time of admission and died of respiratory and circulatory failure or hepatic coma after only one course of chemotherapy or without chemotherapy. The remaining 15 patients received a multidrug combination regimen of 2–22 courses of chemotherapy, with an average of 8.9 courses. The main chemotherapy regimens were 5-fluorouracil (5-FU) based multidrug combinations ((5-FU + vincristine (KSM) + vincristine (VCR) or 5-FU + KSbf + abscisicidal mustard (ATl258) + VCR) or the EMACO regimen.Five patients received 1–5 courses of 5-FU brain perfusion in conjunction with systemic chemotherapy, with an average of 3.4 courses. The average number of sessions was 4. The interval between each session was 2-3 d.Among the patients who received more than 2 courses of chemotherapy, the 5 patients with brain metastases received subarachnoid puncture intrathecal MTX at 10–15 rag each time, 3-4 times a course. 17 courses of MTX were given intrathecally to the 5 patients, an average of 3.4 courses per patient.Tube chemotherapy or embolization: in five cases, the uterine artery was cannulated six times for uterine lesions, one case was cannulated for suspected renal metastases, one case was cannulated for hepatic metastases, and one case was embolised with bilateral supracerebral, uterine, and internal iliac arteries for hematuria with bleeding from vaginal metastases.Twelve of the 19 cases were treated with a total of 15 different procedures. Four cases underwent total hysterectomy with double adnexal resection and high ligation of both ovarian arteries, and one case underwent simultaneous resection of a brain metastasis and brain repair. The remaining five cases were treated surgically at outside hospitals. Two cases underwent dissection, two cases underwent craniotomy, and one case underwent total hysterectomy + right fallopian tube + part of right ovary + part of small intestine + appendix.Treatment effect: the clinical treatment effect is evaluated according to the efficacy evaluation criteria of solid tumors, including four criteria: complete remission (CR), partial remission (PR), no change (SD), and progression (PD), and the effective rate is PR and CR.Analysis of influencing factors: gender, number of metastases, RPA classification, MMSE score before radiotherapy, extracranial metastasis, and other factorsAnalysis of toxicity and adverse reactions: including hematological toxicity, leukopenia, thrombocytopenia, gastrointestinal toxicity, nausea and vomiting, and diarrhea, except for drug-resistant patients, the main manifestation of drug resistance is the enlargement of lesions by more than 50% compared to pretreatment or the appearance of new distant metastasesQuality of life assessment: the SF-36 scale was used to evaluate patients' “social, emotional, cognitive, and spiritual functions. The total score of each function was 100 points, and the score was directly proportional to the patients' quality of life.t-test. P < 0.05 has statistical significance as GraphPad Prism8.The collected data were entered into the Excel table, and statistical SPSS 22.0 software was used to analyze the data. If the collected data met the normal distribution, composition ratio, and rate description of count data, measurement data were expressed as (mean ± standard difference) and Of the 19 patients, four were admitted to the hospital and died of liver coma or respiratory and circulatory failure without chemotherapy or after only one course of chemotherapy because they were nearing the end stage of metastasis in multiple organs. Out of the other 15 patients, 11 patients achieved complete remission after 2–22 courses of chemotherapy, 2 patients survived with tumours in their lungs after normalisation of their blood biochemical parameters, and 2 patients were discharged after 7 and 14 courses of chemotherapy, respectively. The complete remission rate was 73.3% (11/15). The 11 patients in complete remission were followed up regularly after discharge, the shortest being 4–43l-months and the longest being 6 years, with no signs of recurrence. Two patients with residual lung tumours survived, one for 8 years and the other for 6 months, with normal blood 13-HCG.The objective remission (ORR) rate was 74.1% (23/31), with a CR rate of 19.3% (6/31) and a PR rate of 54.8% (17/31) .st August 2016, of the 31 patients, 16 had intracranial progression, 8 had progression of the original lesion, 5 had new lesions, and 3 had progression of the original lesion with new lesions, of which 7 were treated locally with gamma knife, 3 were treated surgically, and the rest were treated symptomatically. Out of the 23 deaths, 5 were due to brain herniation due to intracranial progression and the rest to systemic failure due to combined liver, bone, and other systemic metastases. The median time without intracranial progression was 11.5 months, the median survival time was 13 months, and the 1-year PFS and OS were 63.1% and 64.5%, respectively  in 25 of the 31 patients before enrollment were significantly improved in 12 patients and slightly improved in 8 patients after treatment, resulting in a neurological remission rate of 80%. As of 1P < 0.05) (P < 0.05). Multifactorial analysis showed that preradiation MMSE score (27–30 vs <27) was an independent prognostic factor for PFS and RPA grade (grade 1 vs. grade 2) and was an independent prognostic factor for OS  . Sex, nur for OS .Toxic adverse reactions during the administration of TMZ were mainly hematological and gastrointestinal. No liver, kidney, or cardiac related toxicity was observed in this group and there were no treatment-related deaths .After treatment, the social, emotional, cognitive, and spiritual functions increased significantly in both groups, as shown in Patients with metastatic brain metastases from choriocarcinoma or erosive staphyloma often have clinical symptoms such as hematuria and difficulty in urination. In combination with ultrasound and other imaging studies, early detection and treatment are often possible.In our group of 19 patients, apart from 4 brain metastases who died of severe disease, the remaining 15 patients were treated with complete remission in 11 cases, partial remission in 2 cases, and progressive disease in 2 cases. Most of the patients were treated with 5-Fu-based combination chemotherapy  or EMA/CO regimens. To enhance the efficacy, 5-FU cerebral perfusion can be administered concurrently during or before each course of treatment for brain metastases, with a reduced amount of systemic intravenous dosing. In 5 of the 19 cases in this group, 5-FU brain perfusion was administered in parallel with systemic chemotherapy. 5-FU brain perfusion can maintain a higher concentration of the drug in the brain while keeping the total amount of 5-FU virtually unchanged, resulting in better efficacy and fewer adverse effects. The treatment of renal metastases, once clearly diagnosed in the early years, was usually surgical removal of the affected kidney. Since 5-FU chemotherapy has proved to be effective, chemotherapy alone has been used for those with less severe bleeding. However, if severe bleeding occurs, the kidney is also removed and postoperative chemotherapy is given.In this group, two patients had both renal and brain metastases, of which one was in complete remission and one died. The prognosis of brain metastases was better than that of kidney metastases, which was considered to be related to the following factors. (1) Differences in disease: the prognosis of brain metastases is not uncommon in gestational trophoblastic tumours and the incidence of brain metastases has been reported in the literature to range from 8% to 28% BJ. Once brain metastases occur, the condition is more dangerous and is one of the main causes of death. The prognosis of brain metastases combined with liver metastases is the worst, and none of the 10 cases of brain-hepatic metastases in this study survived ∞ J. Another study reported that the 5-year survival rate of patients with liver-brain metastases was only 10%. In this study, there were no cases of brain metastases combined with liver metastases. (2) Patients with brain metastases who received systemic chemotherapy and 5-FU cerebral perfusion had a significant effect. Four of the patients with brain metastases were cured by 5-FU cerebral perfusion, while one case had combined brain and kidney metastases. The 5-FU cerebral perfusion can maintain a high local concentration of the drug in the brain while keeping the total amount of 5-FU virtually constant .The primary focus of malignant trophoblastic tumours usually originates in the uterus and develops outward from the uterus after a certain period of time . After aMost chemotherapeutic drugs are only moderately active in brain metastases, with nitrosoureas, temozolomide, topotecan, and leucovorin showing high concentrations in the brain. In breast cancer, small-molecule targeted drugs such as neratinib in combination with capecitabine also showed good ORR for intracranial metastases, and large-molecule monoclonal antibodies such as T-DM1 also showed some efficacy, probably due to heterogeneous leakage of the blood-tumor barrier . In termResearch on brain metastases has never stopped, and new compounds with higher molecular specificity, including higher blood-brain barrier and blood-tumor barrier permeability, are being developed. Strategies to increase drug delivery across the blood-brain barrier are also being investigated, including opening the tight junctions of the blood-brain barrier, inhibiting the blood-brain barrier efflux pump system, optimizing drug selection, and optimizing drug delivery . The stuIn conclusion, brain metastases from trophoblastic tumours can be treated with whole-body chemotherapy based on 5-FU or EMA/CO regimens, supplemented by local infusion of 5-FU into the brain. The main route of metastasis for renal metastases is blood transfer, whereas brain metastases may be mainly from local infiltration. Patients with renal metastases have a poorer prognosis than those with brain metastases. In the event of hemorrhage from brain metastases, selective arterial cannulation and embolization may be preferred to avoid incomplete surgery and a range of postoperative complications that may arise from emergency open surgery. After treatment, the social, emotional, cognitive, and spiritual functions of the two groups increased significantly. The results fully confirm that chemotherapy treatment for patients with malignant trophoblastic tumors brain metastatic can improve the quality life of patients can feel the warmth of family, and better return to society, which is of great significance to the improvement of patients' quality of life.The present study excluded the influence of confounding factors such as primary and metastatic foci on the results. Although PFS and OS were significantly longer in this study compared to other studies, because it was a single-centre, prospective, single-arm study, future multicentre, prospective, randomised controlled clinical studies are needed to understand the effect of treatment of brain metastases from zygotic tumours and the prognostic risk factors during pregnancy.


This research introduces a readily available and non-chemical combinatorial production approach, known as the laser-induced writing process, to achieve laser-processed conductive graphene traces. The laser-induced graphene (LIG) structure and properties can be improved by adjusting the laser conditions and printing parameters. This method demonstrates the ability of laser-induced graphene (LIG) to overcome the electrothermal issues encountered in electronic devices. To additively process the PEI structures and the laser-induced surface, a high-precision laser nScrypt printer with different power, speed, and printing parameters was used. Raman spectroscopy and scanning electron microscopy analysis revealed similar results for laser-induced graphene morphology and structural chemistry. Significantly, the 3.2 W laser-induced graphene crystalline size  is higher than the higher power  formation due to the surface temperature and oxidation. Under four-point probe electrical property measurements, at a laser power of 3.8 W, the resistivity of the co-processed structure was three orders of magnitude larger. The LIG structure and property improvement are possible by varying the laser conditions and the printing parameters. The lowest gauge factor (GF) found was 17 at 0.5% strain, and the highest GF found was 141.36 at 5%. In advanced manufacturing, 3D printing has gained a lot of attention in the last decade. Due to the versatility in the materials, designs, and ease of custom tailoring, products and performing topographies could not be produced using conventional subtractive manufacturing ,2. In reSignificantly, thermoplastic polymer utilization in a fused deposition model (FDM) demonstrated rapid, simple fabrication at an affordable cost; however, the final product could have been executed with poor mechanical, electrical, and thermal properties. Due to these reasons, the current studies have adequately introduced thermoplastics, which exhibit the prevailing mechanical, electrical, thermal, and required properties. However, introducing nanofiller in the polymer matrix has been considered to be a complex process under different conditions, where the major causes, such as adhesion, orientation, and dispersion of the nanomaterials, constrain it. Consequently, this also affects printing parameters and appropriate printing structures.Nowadays, several studies have been introduced with laser-induced techniques; similarly, laser-induced graphene (LIG) has been used for multifunctional applications. Reportedly, the use of the laser process offers a top-down, noncontact method that produces a graphene-like structure through the laser irradiation process . For ins−1, respectively. However, the intensity peaks for performed laser power samples at 10, 20, and 30% were not very high. Moreover, the D and G bands were connected, corresponding to high-temperature annealed nano graphite. The best result was achieved at 40% laser power, which was used to determine the piezo resistivity of the strain sensor [Recently, Tavakkoli et al. reported a similar kind of investigation with a single laser pass, and the study was performed with 10–40% of laser power from a 30 W laser . The laser spot size was 76 μm, and the scan rate was 2.54 cm/s). All samples indicate D and G bands at 1300 and 1550 cmn sensor , as oppoA high-precision 3Dn-300 nScrypt printer fed with the Ultem filament with 1.75 diameters  extruded via an nFD material extrusion tool head was used to fabricate all specimens in this study . Before 2 laser with a wavelength of 10.6 μm m was helpful in generating the graphitic sensor arrays through the irradiation on the printed specimen film. The beam size of the CO2 laser was ~100 μm, and the thickness of the specimen was 0.3 mm. The laser beam inscribed lines on the printed surface during the process, which was designed via CorelDRAW X3 graphic design software. Pulse per inch or pixel per inch (PPI) and laser speed were fixed at 500 PPI and 38.1 mm/s, respectively. Furthermore, the power (3.2–4.0 W +/− 0.2) was varied effectively to understand the laser-power-dependent changes. Hence, the changes in morphology, electrical properties, and heat transfer were investigated; however, the laser parameter was selected based on relevant studies [Initially, the printing temperature of the nozzle (350 μm) and the bed were kept at 390 °C and 160 °C, respectively. A laser engraving and cutting system  equipped with a CO studies ,32.aL) was calculated from the utilized Raman wavelength and the ratio of the integrated intensity G/D peaks. Equation (1) was used to obtain aL:The Raman spectroscopy (Renishaw inVia Raman microscope) analysis was performed with a 633 nm excitation laser at 1 mW. In addition, Ultraviolet–visible (UV-Vis) absorbance and transmission were measured using the UV-5000 UV-Vis NIR spectrophotometer (Varian). Significantly, the crystalline size along the axis (The wavelength used in this study (2 laser irradiation) morphological structures were examined with scanning electron microscopy (SEM) (Phenom XL Desktop SEM at 10 kV). Probes were employed to evaluate the resistance of the LIG lines recorded by a multimeter. The resistivity was calculated using the cross-sectional values of the LIG from SEM and the length of the graphitic line. For all tests, a silver paste (Electron Microscopy Sciences) and a gold conductive tape were applied on the edge of the sensor for connection then cast with an epoxy resin layer onto the LIG surface for compaction to avoid delamination from the PEI surface. Further, a cyclic three-point bending test was conducted on the sample using an AGS-X mechanical test machine , and the following settings were used for the test in detail: gauge length = 20 mm; displacement rate 4 mm/s; and cyclic strain 0.5, 1, 3, and 5. At the same time, the change in resistance of the mechanically deformed sensor was recorded by a Keithley 2401 source meter controlled by a homemade LabView user interface. Furthermore, the gauge factor was used to investigate the sensitivity of the LIG-PEI corresponding to the strain, the gauge factor (Equation (2)),Then formed graphitic line features’ , 1600 cm−1 (G-band), and 2700 cm−1 (2D bands). The D-band indicated the sp3 atomic state, which denoted few disorders or impurities in the graphene structure; it also suggested an amorphous carbon component [−1 indicate the few-layered graphene stacks [COomponent ,34. Nexte stacks . LIG derIn addition, the relationship between the laser power and the intensity ratio of the G to D peaks was calculated. Additionally, the statistical analysis of IG/ID was determined at five different points and plotted against the laser power in Here, the UV-VIS analysis was investigated to estimate the light–matter interaction of the printed PEI tab and LIG scraped from the surface, as shown in Two absorption peaks were noticed in The optical images in The surface roughness was determined by comparing the LIG on the PEI at low and high laser power. Compared to the low power shown in The calculated resistivity maintains a slow steady increment as the laser power increases from a low value to 3.8 W, as shown in Altogether, the extensive analysis and property observation of material and additive manufacturing introduces the performed material into sensor application. Owing to that, the potential performance of LIG as a sensor of electronic components was investigated via piezo resistivity tests. The 3D printed LIG-PEI sample was utilized as a strain sensor, measured with the bending of deformed resistance changes. Since the PEI is a plastic material, a plastic deformation was expected, where considerable strain deformation exhibited a variation of the relative resistance change.Based on previous studies, 3.2 W induced LIG-PEI was used for the bending test, which was carried out with four different strains ; bending–releasing cycles were demonstrated. The change in the relative resistance is shown in Overall, this work exploring co-processing and fabrication methods helps to improve the manufacturability/design, repeatability with lesser defects, and scalability of a material. It also emphasizes (1) in situ processing for deposition and irradiation, (2) precision deposition of 350 microns, and (3) combinatorial surface characterization and roughness to explain the improved performance of the processed strain sensor. The demonstrated unique ability of the printable polymer material (using the fused filament fabrication AM technique) with certain desired thicknesses, orientations, and techniques is readily available for further processing (known to be laser processing) without any chemical modification or surface treatment. In addition, the characterized piezo resistivity of the LIG was helpful in understanding the process–structure–property relationship . SignifiIn summary, a 3D-printing technique has been used with laser processing as a hybrid processing and co-production method to fabricate flexible, innovative components and for the purpose of sensor-monitoring deformation. The formation of graphitic structures can be prepared on the surface of 3D-printed PEI by controlling the laser irradiation conditions. The morphology and piezo resistivity of the LIG were studied to establish the related process–structure–property relationship. The LIG-PEI sensor shows high sensitivity when subjected to the mechanical test. It is demonstrated as a strain gauge for printable structural self-monitoring flexible sensors of polymeric composite. As shown in this work, the unique performance of LIG using the AM fabrication technique with laser processing can be highly valuable in different industrial sectors for producing customized printable, on-site, and flexible electronics.


Camellia sinensis (Fuzhuan tea). In this study, we examined differences in the bacterial community structures of loose, lightly-pressed, hand-made, and machine-pressed Fuzhuan teas and raw dark tea. We observed notable differences in the bacterial communities of the five groups, where there were only 51 consensus sequences. ASV/OTU Venn diagram, Chao1, Ace, Simpson indices, and dilution curve analyses consistently revealed that machine-pressed tea exhibited the highest bacterial diversity. Taxonomically, Actinobacteria, Firmicutes, Proteobacteria, and Cyanobacteria were the dominant bacterial phyla in each group, whereas Corynebacterium, Methylobacterium, and Bifidobacterium were the dominant genera. Our findings revealed significant differences in the bacterial community structures of different Fuzhuan tea products derived from the same raw material, with bacterial diversity rising with increased product compaction.The composition and diversity of microbial communities are of considerable significance to the quality development of  This teackaging ,3 and anackaging , blood lackaging , liver packaging , and immackaging ,8 functiFungal fermentation during the processing of Fuzhuan tea is considered to be the key factor contributing to its unique flavour and properties . In receThe rapid development of the Fuzhuan tea industry has advanced its processing technology and facilities, superseding the traditional fermentation technology with modern fermentation. Also, different types of processing technology have evolved such as bulk, lightly pressed, hand-made, and machine-pressed Fuzhuan tea. Naturally, these processing techniques contribute to creating different microbial communities in Fuzhuan tea which in turn influences its quality attributes . Consequ22.1Tea samples were collected from a production workshop of Yiyang Guan-Longyu Dark Tea Development Co., Ltd, Hunan Province, China, on 5 March 2021. The samples included raw dark tea (G0), loose Fuzhuan tea (G1), lightly-pressed Fuzhuan tea (G2), hand-made Fuzhuan tea (G3), and machine-pressed Fuzhuan tea (G4) and were derived from the same batch of raw dark tea material. Among these, G1 was loose tea, while G2, G3, and G4 were pressed teas, with the pressing degree gradually rising from G2 to G4. All the samples were analysed in three biological replicates.2.2Genomic DNA was extracted from the samples, using the Soil DNA kit , with the purity and concentration of the extracted DNA determined using agarose gel electrophoresis. Aliquots of the purified DNA were diluted to a concentration of 1 ng/μL with sterile water and stored at −80°C for later use . The totUsing DNA extracted from G0, G1, G2, G3, and G4 samples as templates, we used the primer pair 338F (5′-TCCGTAGGTGAACCTGCGG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) to amplify the V3–V4 variable regions of bacterial 16S rRNA gene sequences.The PCR reaction mixtures contained 10 ng of DNA template, 2.5 μM upstream and downstream primers, and 2.5 μM DNA polymerase , made up to a final volume of 30 μL with PCR buffer. The PCR amplification conditions were as follows: pre-denaturation at 98°C for 60 s, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s, with a final extension at 72°C for 5 min. PCR products were detected using 2% agarose gel electrophoresis. The samples were mixed at equal concentrations according to the PCR product concentration, and the PCR products were detected using 2% agarose gel electrophoresis after thorough mixing.2.3Having qualified the constructed library based on Qubit quantification and library detection, we obtained an original data file by using the Illumina (MiSeq pe300) platform for sequencing, with the data being converted to sequenced readings after base calling. The results were stored in FASTQ format, containing information on readings and corresponding sequencing quality.2.4The original data were spliced and filtered to obtain clean data. Based on the valid data, we performed sequence denoising or operational taxonomic unit (OTU) clustering, using the process of QIIME2 dada2 analysis  or VsearAmplicon sequence variants (ASVs) or OTUs were analysed to obtain information on within-sample species richness and evenness . Multi-sThe number of ASVs/OTUs per sample was obtained using QIIME2, and the numbers of common and unique ASVs/OTUs among the samples were displayed using Venn diagrams . The sam33.1The number of valid tags in the five groups of the samples was more than 53,000, with the Q20 and Q30 values greater than 95.54 and 88.58, respectively. More than 29,000 high-quality sequences were obtained. For each sample, we obtained more than 270 ASV/OTU sequences, with an average data quality of 35 or higher. For all the samples, the sequencing quality met the requirements for subsequent analysis.3.2The Venn map was constructed to depict the similarity in the bacterial populations of the different samples . Among t3.3Values obtained for the Alpha diversity Chao1, Ace, and Simpson indices of each sample are shown in The Simpson index is mainly used as a measure of species diversity. The lower the Simpson index value is, the higher the species diversity of a sample is. As shown in 3.43.5The results of PCoA analyses of the five groups are shown in 3.6The results of the taxonomic annotations are shown in Actinobacteria, Firmicutes, Proteobacteria, Cyanobacteria, Bacteroidetes, Verrucomicrobia, Fusobacteria, Synergistetes, Deinococcus-Thermus, Planctomycetes, Patescibacteria, Tenericutes, Chloroflexi, Spirochaetes, Nitrospirae, Gemmatimonadetes, and Acidobacteria, the top ten of which are shown in Actinobacteria was identified as the predominant bacterial phylum, followed by Firmicutes, Proteobacteria, and Cyanobacteria. This was consistent with the results of MiSeq sequencing analysis of dark tea products, such as Fuzhuan tea and pu-erh tea, reported by Fu et al. [Firmicutes and Actinobacteria to be the dominant phyla.At the phylum level of classification, we annotated 17 phyla with clear status among the Fuzhuan tea samples subjected to the different processing techniques; namely, u et al. , who fouActinobacteria, Firmicutes, and Bacteroidetes in G1 rose by varying degrees when compared to those in G0. The numbers of Proteobacteria and Cyanobacteria fell in G1 compared to those in G0. Compared to those in G0, the numbers of Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes in G2 increased by a different degree, whereas there was a significant reduction in the number of Cyanobacteria. The pattern of phyla was found to be essentially the same between G3 and G1. Only the number of Cyanobacteria significantly increased (1.48 times), while the numbers of Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes decreased in G4 when compared to G0. These observations verified that the different processing techniques significantly influenced the structures of the bacterial communities in Fuzhuan tea.With respect to the top five predominant phyla, the numbers of Corynebacterium, Methylobacterium, Bifidobacterium, Faecalibacterium, Escherichia–Shigella, Actinomyces, Bacteroides, Collinsella, Sphingomonas, Prevotella, Candidatus Xiphinematobacter, Caldicoprobacter, Aminobacterium, uncultured Acidothermaceae, Leptotrichia, Coprostanoligenes group, and Veillonella were detected commonly. The number of species in each group at the genus level is shown in Corynebacterium was most predominant, followed by Methylobacterium and Bifidobacterium. Compared to G0, a varying degree of increased numbers of Corynebacterium, Faecalibacterium, Escherichia–Shigella, Actinomyces, and Collinsella was detected in G1, whereas a reduction was observed for all the other genera, among which, a significant (approximately 10%) reduction in the number of Methylobacterium was found. Increases to a different extent in the numbers of Corynebacterium, Faecalibacterium, Escherichia–Shigella, and Bacteroides were observed for G2 when compared to G0, whereas the numbers of all the other genera declined during the processing. The varying degrees of increases in the numbers of Corynebacterium, Faecalibacterium, and Sphingomonas were found in G3 when compared to G0, with the numbers of the remaining genera declining, among which the number of Methylobacterium significantly fell by approximately 10%. Only the number of Collinsella increased in G4 compared to G0, with the numbers of all the other genera decreasing, in particular, the numbers of the unidentified genera. Collectively, these findings highlight the significant effects of the Fuzhuan tea processing techniques on the bacterial community structure, with important implications for the final tea products.At the genus level, we annotated 532 genera with clear status in the different samples, among which, To further study the phylogenetic relationship among the species at the genus level, we used MEGA 5 to obtain representative sequences of the top 100 genera based on multiple sequence alignment . The res4Klebsiella, Lactococcus, and Bacillus play a leading role in the fermentation process of Fuzhuan tea as well as participate in the generation of a large number of flavour compounds in the tea [Aspergillus, Candida, Debaryomyces, Penicillium, and Saccharomycetales significantly contribute to the formation of the unique aroma of Fuzhuan tea [Aspergillus niger, Aspergillus pallidofulvus, Aspergillus sesamicola, and Penicillium mangini can transform theophylline into theophylline in pu-erh tea, thus affecting its quality [The flavour, colour, and aroma attributes of fermented teas and control over them primarily depend on the activities of microorganisms associated with the fermentation process. A better understanding of the composition of this microbial community is required to improve tea product quality . Previou the tea . Aspergihuan tea . Aspergi quality . Chen et quality  reportedIn this study, using Illumina MiSeq technology, the bacterial structure was analysed in the finished products of the differently processed Fuzhuan teas. Seven phyla, 23 classes, 41 orders, 154 families, 532 genera, and 231 species were detected. Among the five groups analysed, only 51 consensus sequences were identified. Among the four groups , G3 and G4 had the lowest (513) and highest (946) numbers of unique sequences, respectively. Alpha diversity index, dilution curve, rank abundance curve, and PCoA analyses consistently pointed to the significant influence of the different processing techniques on the structure of the bacterial communities in the finished Fuzhuan tea products, with the machine-pressed tea (G4) having the highest bacterial species abundance and diversity.Actinobacteria dominated the phyla level, followed by Firmicutes, Proteobacteria, and Cyanobacteria, whereas the genera were dominated by Corynebacterium, followed by Methylobacterium and Bifidobacterium. Some of these bacteria, such as Methylobacterium and Bifidobacterium, were loosely associated with the quality of fermented tea [Faecalibacterium in tea was associated with the regulation of the intestinal tract [Actinobacteria helps to improve the quality of beverages and baked goods [Corynebacterium can enhance the concentration and taste of tea soup [We also detected significant differences among the different tea products with respect to the number of ASV/OTU sequences annotated at the levels of kingdom, phylum, class, order, family, genus, and species. G4 had the highest number of ASV/OTU sequences annotated to each level. nted tea . The preal tract . Xynanased goods . Amino atea soup .In the present study, the differences in the bacterial community structure among the different types of Fuzhuan tea were attributed mainly to the differences in the compression degree of the raw materials during the processing. Unlike loose Fuzhuan tea, not pressed, lightly-pressed, hand-made, and machine-pressed Fuzhuan teas, pressed with a progressive degree, damaged the tea tissues to a different extent, thus changing the releases of the contents of the leaf cells. This in turn plays an important role in influencing the subsequent fermentation. The results consistently indicated that the machine-pressed Fuzhuan tea contained the highest abundance and diversity of bacterial species. In other words, the excessive releases of intracellular tea contents significantly promoted the growth and development of bacteria. The degree of compaction among the four processed teas progressively rose from G1 to G4. Given the difference in compactness, the differently processed teas were characterized by different internal air circulation and humidity which in turn affected the growth of bacteria, and thus, the bacterial community structure. Overall, the change in the bacterial community structure during the fermentation of Fuzhuan tea significantly depended on the product morphology.In this study, Illumina MiSeq sequencing was used to provide insights into the structure and diversity of the bacterial communities of the differently processed Fuzhuan teas. However, the complex nature of these tea communities did not allow for the annotation of all the detected ASV/OTU sequences to specific species, which warrants further analyses. An additional focus in future is required on the specific metabolic mechanisms of bacterial community members with respect to the development of the quality attributes of Fuzhuan tea, such as their respective contributions to flavour formation. Given these insights, it may be feasible to control the different processing conditions to fully exploit the biotransformation properties of the tea microbiota. This in turn may enhance the quality of Fuzhuan tea, and thus, raise its manufacturing technology as well as quality control and assurance management to a new level.


Background: Unhealthy lifestyles are strongly entrenched in healthcare universities and have sometimes been linked to stress or lack of sleep. This study investigated the prevalence of toxic habits , stress levels, perceived health status, and sleep duration and assessed the connections between toxic habits and said well-being measures, as well as healthcare students’ perception of the influence of the COVID-19 pandemic on these health-related behaviors. Methods: In a cross-sectional study, healthcare students from Alfonso X University (Spain) completed a health survey composed of Alcohol Use Disorders Identification Test (AUDIT-C), Perceived Stress Scale (PSS-10), self-perceived health status, and the number of hours of sleep. Results: A total of 997 healthcare students completed the survey, of which 982 were analyzed. Being a smoker (32.2%) was associated with worse health status and insufficient sleep. Risk drinkers (33.2%) were associated with being female, and the consumption of cannabinoids (6.7%), with being male. These three toxic habits were related to each other. High levels of stress (28.2%) were correlated with worse ratings in the perception of health status (29.2%) and with insufficient sleep (45.8%), and all of them were associated with the female sex. Respectively, 49.3% and 44.2% of students recognized a worsening in their perception of stress and their sleep habits during the pandemic. Conclusion: Healthcare universities must carry out health promotion programs for stress management, sleep habits, and unhealthy lifestyles. Numerous studies have linked unhealthy habits, such as smoking, alcohol, or other drug use, with the development of non-communicable diseases, such as cardiovascular, renal or respiratory diseases, diabetes, cancer, and dementia, all of which are responsible for 70% of deaths worldwide ,2.Preventing these modifiable risk factors in the general population is the direct responsibility of healthcare professionals ,4, and tWe must consider that future healthcare providers are university students who are mostly in the stage of adolescence or early adulthood, where important milestones occur, such as leaving the family home, increased individual autonomy, and a greater desire to experience sensations and be accepted in their peer group. Thus, they are a vulnerable population for the acquisition of unhealthy behaviors  and coulKnowing that the lifestyles that are acquired in this stage will likely drastically influence those that develop during adulthood ,11 and tIn addition to harmful substances, two other determinants that directly influence the health of the college population are stress and sleep duration . Severe Stress in the university population is associated with poorer physical health, poor academic performance, and numerous mental illnesses, such as depression, anxiety, anorexia, or substance abuse, among those who suffer from it . On the In addition to being related to health, the stress in the university population was correlated by some authors with the consumption of toxic substances, objectifying that the most stressed university students presented a higher prevalence of smoking or alcohol abuse ,19,21. OAnother important measure related to well-being and health is sleep duration, whose alteration is related to different physical and psychiatric illnesses . A sleepSince the moment when the WHO declared COVID-19 infection a global pandemic in March 2020, its consequences have been objectified in all spheres of society, including changes in lifestyles and increased levels of stress, both in the general population ,28 and eAnother group vulnerable to worsening well-being during the pandemic is university students , specifiIt has also been studied how the pandemic caused changes in drinking habits in the general population. In a large European study carried out during the first months of the pandemic, a decrease in alcohol consumption in the adult population was observed in 19 of the 21 European countries investigated . In contWe also found contradictory results in the university population. Authors such as Du et al.  and JaffThe aim of this study was to define the prevalence of toxic habits , stress levels, perceived health status, and sleep duration among healthcare university students, as well as to evaluate the connections between toxic habits and said well-being measures. Finally, we wanted to investigate the students’ perception of the influence of the COVID-19 pandemic on these health-related behaviors.A cross-sectional unicentral study was carried out on a population of university students with degrees related to healthcare at Alfonso X el Sabio University (UAX) in Madrid (Spain).Non-probabilistic convenience sampling was used in this investigation. The recruitment criteria were students aged at least 18 years and registered during the academic year of 2021–2022 in one of the academic careers related to healthcare offered by UAX . Data were collected between November and December 2021.This study was conducted through an anonymous survey that was self-completed on an online platform by the student when attending an in-person class. The researchers informed the participants about the study’s objectives and the confidentiality that would be maintained in the data collected throughout the study. Their participation was voluntary, and they did not receive any reward for participating.The study was conducted following the Declaration of Helsinki, and the protocol was approved by the Clinical Research Bioethics Committee of Alfonso X el Sabio University (Resolution 2021_10/099). All participants gave their informed consent before participation, knowing that they could revoke their collaboration during the investigation.The survey (Annex 1) was based on the Spanish National Health Survey , in assoThe survey included age, sex, academic major and year, and diverse socioeconomic features .Smoking habit: This was defined as non-smokers, active smokers, or former smokers, who were those that had remained abstinent for at least the last 6 months. For establishing risk ratios, the sample was aggregated into smokers and non-smokers. Smokers were also asked about their intention to quit the habit. Finally, exposure to tobacco smoke was also investigated.Alcohol consumption: The alcoholic habit was determined according to the frequency of alcohol consumption, classifying the participants as habitual drinkers if they declared consuming alcoholic beverages at least once a week, occasional drinkers if their consumption was less than once a week, or non-drinkers.The Alcohol Use Disorders Identification Test (AUDIT-C) was used to detect problems due to alcohol abuse; it consists of 3 questions on the consumption habit and is adapted from the longer AUDIT questionnaire . The scoDrug consumption: It was quantified by the answer to the following question: “How often do you consume any of the following substances: Tranquilizers, cannabis, cocaine, ecstasy, hallucinogens, or amphetamines?” The possible answers were “never”, “occasionally”, “weekly”, or “daily” for each of two time periods (“throughout your life” and “within the last 30 days”), following those reported in other studies .Stress perception: This was assessed using the Perceived Stress Scale-10 (PSS-10) , which wSelf-assessment of health status: This measure was used as a global health indicator  and was Sleep duration: This was also assessed by determining the mean number of hours of sleep, considering an insufficient sleep duration when it lasted less than 7 h, as defined by other authors ,51.As Du et al.  investigData analysis was performed using SPSS Version 22.0 .Quantitative variables following normal distribution were defined using mean and standard deviation. Discrete variables were defined using some cases and percentages.t-test and the ANOVA test were used. A p-value < 0.05 was considered statistically significant.For the comparison between qualitative variables, the chi-square test was used. When the expected value was less than 5 in any of the boxes of the contingency table, it was necessary to use Fisher’s exact test. The magnitude of the association was estimated using the Relative Risk (RR) ratio and expressed with a 95% Confidence Interval (CI). For the comparison of independent quantitative variables, Student’s The total population consisted of 2059 students, of whom 997 gave their consent to participate in this research study and completed the questionnaire. Fifteen participants were excluded due to incomplete surveys. Thus, data from 982 students were analyzed, including 652 females (66.3%) and 330 males (33.7%), with a mean age of 20.5 ± 3.8 years. Of the total sample, 854 (86.9%) were Spanish students, and 128 (13.1%) were foreign students. The academic major of the students and the years are described in The socioeconomic features of the sample are described in A total of 64.3% of participants were non-smokers, while 32.2% (316) were active smokers, and 3.6% were former smokers. Of the active smokers, 120 (38%) failed an attempt to quit smoking, whereas 286 (90.6%) seriously intend to quit in the next months. Of the total sample, 20.1%, 28.7%, and 68.7% were exposed to tobacco smoke at home, inside the university campus, and in entertainment venues, respectively.A total of 872 (88.7%) subjects drank alcoholic beverages, 57.8% were habitual drinkers, and 30.9% were occasional drinkers. Only 35.3% of the students reported drinking alcohol daily, whereas 74.1% drank alcoholic beverages during the weekend. According to the AUDIT-C classification, the sample’s prevalence of risky alcohol consumption was 33.2%, and 21.7% frequently binge-drank (at least monthly) .In the last 30 days, students had consumed the following drugs at least once per week: 5.6% benzodiazepines, 6.7% cannabinoids, 0.5% cocaine, 0.3% ecstasy, 0.2% amphetamines, and 0.1% LSD. The frequency of drug intake is shown in the following table .p < 0.001). Similarly, active smokers were also associated with binge drinking habits ; p < 0.001).Dangerous alcohol intake, according to the AUDIT-C definition, was significantly associated with active smokers ; p < 0.001), whereas benzodiazepines and other drugs did not show any significant association with the smoking habit.Active smokers were also significantly associated with cannabinoid use ; p < 0.001) and cocaine ; p = 0.027). In contrast, dangerous alcohol consumption, according to the AUDIT-C criteria, was only significantly associated with cannabinoid intake ; p < 0.001) ; < 0.001) . BenzodiA prevalence of stress perception was obtained and evaluated with the PSS-10, with 11.4% of the subjects presenting low stress, 60.4% medium stress, and 28.2% a high level of stress.The perception of health status during the last 12 months is summarized in Regarding sleep duration, 448 participants (45.8%) acknowledged sleeping less than 7 h, which is considered insufficient sleep. The mean sleep time among all the students was 6.5 ± 2.3 h, with 46.5% of them perceiving it as restful sleep.p < 0.001) and with sleep duration of at least 7 h/day ; p = 0.041). Likewise, such sleep duration was also correlated with lower stress levels (p = 0.003).Good–excellent health status perception was significantly associated with lower stress levels (PSS-10) (p = 0.036).Students exposed to tobacco smoke at home reported a worse health status than those who were not exposed ; p = 0.001).Smokers were associated with insufficient sleep duration (<7 h/day) ) and poorer perceived health status ; However, we failed to demonstrate an association between any toxic behavior analyzed with perceived stress, according to the PSS-10. We were also unable to demonstrate an association between the harmful use of alcohol or other drugs with sleep duration or perceived health status.p < 0.001), there were no significant differences in the prevalence of binge drinking. In contrast, females showed a lower cannabinoid intake habit ; p < 0.001). There were no differences in the rest of the toxic habits according to gender.Despite the fact that 12.9% more females than males showed risky alcohol consumption  and insufficient sleep duration ; p = 0.02) than males.Simultaneously, females reported worse health status ; p < 0.001) ; < 0.001) .p < 0.001) and high-risk drinkers ; p < 0.001) than the rest of the university degrees analyzed.Physical therapy students showed a higher frequency of binge drinking ; Although most respondents (77.2%) considered that their smoking habits had not changed since the year before the COVID-19 pandemic, 2.6% acknowledged having started smoking during that period, and 9.4% said that they currently smoked more often. In contrast, 3.3% quit smoking, and 7.4% reduced their consumption .A total of 55% of the participants recognized that the COVID-19 pandemic did not alter their alcohol consumption, although 22.6% reported an increase in their alcohol intake.When comparing the stress perception during the last 12 months with their previous stress perception before the pandemic, 49.2% of the participants recognized an increase during the pandemic period.This was in turn correlated to the fact that 44.2% of the students identified that they began to sleep worse during the pandemic period.However, similar proportions of students perceived their current health status to be worse (27.7%) and better (30.5%) than in the 12 months before the pandemic.The present study analyzed toxic habits and measures related to well-being in a population of Spanish healthcare students during the second year of the COVID-19 pandemic, when social distancing measures were reduced but still persisted and were constantly changing according to the evolution of the COVID-19 infection.p < 0.001). This finding was already documented in studies conducted with healthcare students [Almost one-third of the sample were active smokers, and two-thirds acknowledged exposure to tobacco smoke in entertainment venues. These figures were in line with studies of other university populations ,53, althstudents ,54, as wstudents . This alstudents .The significance of this finding does not only redound to the health of these young people but also to a likely worse predisposition to promote healthy habits, as was reported by Duaso et al.  in a stuAs a positive finding, most smokers (90.6%) were serious about quitting smoking in the next few months, which reinforces the need to assist students in smoking cessation in schools for future healthcare workers .The prevalence of risky drinking found in this sample (33%) was in line with that reported in other research among Norwegian students 35.9%) , althoug% , althoBinge drinking is associated with multiple negative health consequences, such as an increased development of alcohol dependence in adulthood , among oIn this research study, the most used drug type during the last 30 days was cannabis, with a prevalence of 15.6%, similar to studies such as the one conducted by Reis et al.  in BraziDuring COVID-19 confinement, modifications in toxic habits were objectified among college students, probably related to social isolation, limited access to harmful substances, and health-related reasons . In thatA national survey of the general Spanish population comparing tobacco, alcohol, and illicit drug use between the pre-pandemic period and the first year of COVID-19 showed a decrease in the use of all the drugs analyzed, especially in the 20–24-year age group, which may be due to the recreational use that often justifies their use at this age . These rWhen analyzing the relationships among between toxic habits, binge drinking and hazardous alcohol use formed with tobacco and cannabis use a dangerous combination triangle of addictive substances, which was also found in previous studies ,52. In tThe study of stress in the university population is essential, due to the greater susceptibility it generates to mental health problems . PerceivThe increase in perceived stress was accompanied by a reduction in sleep in almost half of the sample, which was congruent with the direct relationship that was objectified between the two factors in this and other investigations ,38,67.One-third of the students surveyed reported a fair, bad, or very bad health status, which was three times the prevalence found by Bennasar-Veny et al.  in anothInstructing healthcare students in stress management could result not only in improving their sleep quality and health status, but also in helping them to acquire greater competence to provide safe and quality clinical care in the future . This woSome studies have associated the university population with the consumption of harmful substances, such as tobacco and alcohol, with a higher level of stress ,19,21.However, in line with other investigations ,22,23, aIn contrast, smoking, both active and passive, in the student’s home was specifically linked to poorer perceived health status and shorter sleep duration. This was found by Altun , who obsThere were gender differences, as female students presented a significant increase of 12.9% in risky drinking compared with males. This represents a singularity with respect to the rest of the studies consulted in the university population, where the male sex was a risk factor among risky drinkers both in the period before COVID-19  and durip < 0.001) than males, which was consistent with the reviewed studies [In contrast to alcohol consumption risk, the female population consumed fewer cannabinoids ;  studies .Graves et al.  reportedIn line with the relationship detected between higher levels of stress and a worse index of self-perceived health and insufficient sleep duration, we found that gender differences in stress also held true for the other measures of well-being. Gender differences in these health indicators were reported by Benham et al.  in a samFuture research is required to continue to monitor how toxic habits and wellness measures will evolve in the college population once all the constraints brought on by the COVID-19 pandemic are removed.For future research, conducting random sampling and following up on the population with a post-survey could improve the quality of the study.As this was a cross-sectional study, we could not establish causal relationships. Moreover, in this regard, variables such as motivations for consuming toxic substances or the causes of the worsening of well-being measures could have been evaluated, which would have allowed us to analyze gender differences in greater depth. However, as this was a voluntary, self-administered survey, we believe that a longer survey could have led to incomplete surveys. On the other hand, the students had the presence of a researcher while answering the survey, which could have improved the quality of the answers.The use of a self-reported questionnaire as a measurement instrument might have underestimated substance use due to social desirability bias, although these instruments are valid in a university population for analyzing substance use . In addiRegarding the investigation of perceived changes from the period before the COVID-19 pandemic, this study was based on students’ retrospective recall, which could have been affected by recall bias ; thus, tFinally, the participants were mostly women (66.3%) compared with men (33.7%), with proportions similar to those reported in the different studies carried out in university populations .This study revealed a considerable presence of toxic habits, severe perceived stress, and insufficient sleep duration among Spanish healthcare students with a perceived worsening with the COVID-19 pandemic, which highlights the need to implement specific public health educational strategies in the universities of future health providers.The association found between smoking, harmful drinking patterns, and the consumption of cannabinoids implies recommending that these preventive programs treat all psychoactive substances simultaneously.Since severe levels of stress were associated with poorer perceived health status and shorter sleep duration, and the latter two measures with smoking, we suggest that interventions should address multiple behaviors by focusing on stress management, education about habits of sleep, and the immediate consequences of unhealthy habits as pillars to improve the health of students.Worse results were found among female students in all the well-being measures analyzed and in risky alcohol consumption, which leads us to propose that these interventions be carried out with a gender perspective.


The main mechanism of plant tolerance is the avoidance of metal uptake, whereas the main mechanism of hyperaccumulation is the uptake and neutralization of metals through specific plant processes. These include the formation of symbioses with rhizosphere microorganisms, the secretion of substances into the soil and metal immobilization, cell wall modification, changes in the expression of genes encoding heavy metal transporters, heavy metal ion chelation, and sequestration, and regenerative heat-shock protein production. The aim of this work was to review the natural plant mechanisms that contribute towards increased heavy metal accumulation and tolerance, as well as a review of the hyperaccumulator phytoremediation capacity. Phytoremediation is a strategy for purifying heavy-metal-contaminated soils using higher plants species as hyperaccumulators. HoweAnother example is Pb uptake via the endocytic pathway, whereby it is bound to a pectin fraction in the cell wall and then transported to the protoplast ,75. A loRoots are the main organ that accumulates a high metal concentration to avoid damage to reproductive organs (flowers) or photosynthetic disorders (in leaves). To avoid zinc and lead transport to the shoots, Casparian strips in the endodermis block their passage, together with water, into the secretory and vascular tissues. Studies on plants highly contaminated with Pb showed it to be present only in the vegetative plant parts. The ovule and embryo sac, and the seeds developing from them, were Pb-free ,74,76.Alyssum lesbiacum showed a more efficient histidine synthesis, and together with Pb, formed a complex which was transported in the xylem vessels. Such a complex may be significant in terms of increased plant tolerance [However, metal transport to above-ground sections can occur in the xylem. The Ni hyperaccumulator olerance ,76.Plants can maintain metal homeostasis due to transporters contained within the biological membrane . They ar+ exchanger), COPT (specific high-affinity transporter), ABC (ATP-binding cassette), and IREG (iron-regulated transporter) [Transporters are not specific, i.e., in addition to transporting essential/ballast elements in the plant, they transport harmful substances, e.g., too much cadmium, lead, or nickel . Currentsporter) .+ and Cu+, while the other group transports divalent ions, such as Zn2+, Cd2+, Pb2+, and Co2+. P1b-ATPases belonging to the ATPase subfamily that transport Cu2+, Zn2+, Pb2+, Co2+, Mo2+, and Cd2+. They consist of eight transmembrane segments, and an intramembrane motif, that binds metal ions to amine or carboxylic groups and distinguishes P1b-ATPases from other P-type ATPases [A. thaliana, eight metal-transporting ATPases were discovered, namely AtHMA1-8 . Four are Cu+-ATPases (HMA5-8) and three are Zn2+-ATPases (HMA2-4), as well as HMA1, which also transports Cu+; however, the latter’s’ binding site is different from HMA5-8 [Heavy metal ATPases transport substances across membranes using ATP hydrolysis energy . In plan ATPases ,78. In Am HMA5-8 ,78,79.+ to the Golgi apparatus, while HMA6, 8, and 1 transport Cu+ to the chloroplasts. HMA5 is involved in root copper detoxification. Zn2+-ATPases are found in the plasma membrane, and their increased expression has been observed in root and shoot vessels. Excessive HMA4 expression causes increased Zn2+ transport from the roots to the shoots. Considering their distribution, these are involved in metal unloading to/from the phloem, or metal transport to/from the xylem. AtHMA4 also participates in cell detoxification, and in cases of elevated Cd2+ concentration, this transporter removes it from root cells and transports it to the shoots [HMA7 is responsible for transporting Cue shoots ,77,78,79A. halleri, an AhHHMA4 transporter is located in the cell membrane, which participates in cytoplasmic cadmium and zinc removal to outside the protoplast, while AtHMA3 participates in Zn2+, Co2+, and Pb2+ ion sequestration by transporting them to the vacuoles [In vacuoles ,79.A. thaliana, HMA3 increases Cd, Zn, and Co accumulation and tolerance [O. sativa is responsible for Cd transport in root vacuoles. The loss of HMA3 function in A. thaliana and O. sativa reduces cadmium levels in root vacuoles, thereby causing high Cd accumulation in the above-ground sections [The heavy metal carrier ATPase 3 (HMA3) is located in tonoplasts and is responsible for the vacuolar sequestration of metals. In rice, HMA3 limits Cd deposition in seeds and leaves. In olerance . OsHMA3 sections ,78.A. halleri, depend on the HMA4 metal pump, and the increased expression of this transporter in A. halleri is due to a combination of modified cis-regulatory sequences and copy number expansion compared to A. thaliana [Studies on HMA4 have shown that excessive Zn accumulation, and full Cd and Zn hypertolerance in thaliana .2+  and Fe2+  transport [T. caerulescens and A. halleri. The IRT1 transporter, located in A. thaliana, is responsible for metal ion uptake, such as Fe2+, Zn2+, Mn2+, Co2+, and Cd2+ [ZIP transporters are membrane proteins that transport divalent metals and are responsible for maintaining homeostasis by transporting these metals to the protoplast. The ZIP family includes transporters responsible for Znransport ,75. Theiand Cd2+ .2+ transporters in A. thaliana are ZIP4, ZIP9, and ZIP10, while IRT3 is responsible for transporting zinc to the cytoplasm, and ZIP1, ZIP3, and ZIP12 are responsible for intracellular transport through the membranes [Among the Znembranes ,77.ZTN1 and ZTN2) in T. caerulescens, and ZIP6 and ZIP9 genes in A. halleri in nonaccumulating plants, is regulated by Zn level and occurs only in cases of its deficiency, while in hyperaccumulators, the expression of these genes is independent of the Zn level and constantly maintains a high value [A. thaliana, and in ZCAX2 in Z. mays. For the c-1 (occurring on the vacuolar side) and the c-2 (occurring on the cytoplasmic side), loop mutations in their genes inhibit or reduce Ca2+ and Mn2+ transport. They function as filters, i.e., they are responsible for cation uptake or selection. The D-domain is characteristic for the CAX subfamily and is responsible for regulating the transport of calcium and other cations by CAX proteins due to cytoplasmic pH changes [The expression of ZIP genes ) transporter, located in A. thaliana, transports Fe2+ ions from the soil. Meanwhile, AtNRAMP3 ) and AtNRAMP4 ) transfer Fe ions to the vacuole, where the metal deposited in the vacuole can be recovered during germination. Proteins from this family can also transport Cd2+, Zn2+, Mn2+, and Ni2+. For example, the TjNRAMP4 transporter in T. japonicum has the sole responsibility of transporting Ni to the cytoplasm [The NRAMP family of transporters belong to the integral membrane proteins that transport divalent metal ions, mainly Feytoplasm ,81.The NRAMP protein is made of 12 transmembrane helices with a characteristic, long hydrophilic C-terminal of a polypeptide chain, which faces the cell interior .A. thaliana, eight genes have been identified to encode YSL proteins that transport Fe, Zn, and Cu with nicotianamine. The AtYSL2 transporter is located in the leaf cells surrounding the vessels and is responsible for transporting iron complexes from the roots to the vascular bundles. The proteins AtYSL1 and AtYSL3 are responsible for delivering Fe2+ ions to the seeds. AtYSL2 gene expression occurs in cases of optimal iron levels and the stimulation of its expression in the presence of Cu2+ ions. In T. caerulescens, the TcYSL3, TcYSL5, and TcYSL7 genes are responsible for the transport of a complex of metal ions with nicotianamine from the cell wall to the cytoplasm and have been identified in vascular system cells [The YSL proteins belong to the oligopeptide transporting protein family. They are involved in metal uptake and transport, in the form of complexes, in monocotyledonous and dicotyledonous plants. A single oligopeptide chain consists of about 700 amino acids, of which the N-terminal is rich in glutamate and aspartate. In monocotyledons, YSL proteins are located in the roots, where they transport metals, mainly iron in a complex with phytosiderophores. Low iron levels in maize activate phytosiderophore synthesis. In dicotyledons, metals are transported from the apoplast to the cytoplasm in a complex with nicotianamine. Different YSL transporter expressions have been observed in rice and is dependent on the Fe level. In the case of an Fe deficiency, excessive OsYSL2 expression occurs in rice seeds and leaves, and OsYSL15 in roots, while OsYSL13 gene expression is not dependent on this. OsYSL proteins transport iron and manganese complexes with nicotianamine, and their high expression in cells accompany the vasculature, through which they participate in metal transport regulation. In em cells ,81.Z. mays, OsYSL15 in O. sativa, and HvYS1 in H. vulgare. They are responsible for transporting Fe ion phytosiderophore complexes from the soil to the root cells. The second class is responsible for intracellular transport and distribution of metal complexes with nicotianamine or phytosiderophores and includes AtYSL1-4 and 6 in A. thaliana, OsYSL2, 6, 16, and 18 in rice, and HvYSL2 and 5 in barley. Research on a new gene isolated from S. nigrum, SnYSL3, has shown that its expression occurred in cases of excess cadmium and increased over time, but was not induced by the presence of Fe. This may indicate that the SnYSL3 gene does not absorb Fe from the soil. Excessive expression occurred in vascular bundles, but this gene’s transport activity is also significant in other plant parts. The SnYSL3 protein transports a metal–nicotianamine complex and plays an important role in the plant’s stress response caused by excess cadmium [YSL transporters can be separated into two classes. The first is characteristic for Poaceae plants and contains the ZmYS1 transporter in  cadmium .2+, Co2+, Cd2+, Mn2+, and Ni2+ transport. CDF proteins transport metal ions through membranes based on a H+/Me antiport, from the cytosol to the organelles, or outside the cell [A. thaliana, which is responsible for transporting zinc to the vacuole, i.e., sequestration. MTP1 transporters and AtMTP1 homologues have also been found in the cell membrane of the hyperaccumulator T. goesingense. AgMTP1t1 is responsible for Zn2+, Co2+, and Cd2+ transport, and AgMTP2t2 is responsible for Ni2+ transport, which contributes to increased plant tolerance towards these elements. In the hyperaccumulator A. halleri, AhMTP1-3 have been described in the tonoplast, which transport zinc ions to the vacuole. In the Populus trichocarpa x P. deltoides hybrid, MTP1 transporters have been identified which are located in vacuole membranes and are responsible for Zn2+ detoxification [CDF family proteins are responsible for Znthe cell ,75. CDF fication ,74,76. Cfication ,83.2+ ion collection and transport. Five COPT transporters (1–5) have been found in A. thaliana [COPT are responsible for Cuthaliana ,83, withthaliana ,83.N. tabacum. In transgenic plants, IREG transporter activity is high and contributes to an increased Ni2+ tolerance and uptake reduction, as well a sensitivity to, and accumulation of, Pb2+. The most well-known transporter is LTC1 (lipid transfer at contact site 1), which is capable of moving Cd2+, Ca2+, Pb2+, K+, and Na+; it is located in the cell membrane. It detoxifies the plant of cadmium and regulates various cellular processes, with detoxification dependent on soil Ca2+ concentration. When the calcium concentration is low, LTC1 may contribute to an increased root Pb2+ accumulation [IREG transporters are nonselective cell membrane channels, which are permeable to mono- and divalent metal ions. They are homologous to cyclic nucleotide channels in animals. NtCBP4 is one of the IREG family transporters and is located in mulation ,77,84.A. thaliana is responsible for the transport of cadmium ions and its complexes with other substances, and its activity occurs when Cd2+ is present in the plant, or, to a lesser extent, Cu2+ or Zn2+. AtATM3 (ABC transporter of the mitochondria (3)) is also responsible for transporting cadmium ions and its complexes with other substances, e.g., glutamine synthetase with Cd2+. AtPDR8 (pleiotropic drug resistance (8)) is located in the root cell membrane of A. thaliana and is responsible for the transport of Cd2+ and Pb2+ ions, and their complexes, from the protoplast, thus decreasing cytosol metal levels and increasing the plant’s cadmium and lead resistance. AtPDR12 (Pleiotropic Drug Resistance12) is found in the cell membrane of A. thaliana and acts as a pump that transports lead ions and its complexes from the cytoplasm, thus increasing the plant’s resistance [ABC transporters derive energy from ATP hydrolysis by transporting ions, lipids, carbohydrates, peptides, antibiotics, and xenobiotics, among others. They can be classified into (1) the MRP (multidrug resistance-associated protein) subfamily, which are proteins involved in toxin resistance, (2) the ATM (ABC transporter of the mitochondria) subfamily, which carry mitochondrial proteins, and (3) the plant PDR (pleiotropic drug resistance) subfamily associated with pleiotropic toxin resistance. The AtMRP3 transporter identified in the hyperaccumulator sistance ,85.ABC transporters consist of three structural types. The full transporter contains two transmembrane and two nucleotide-binding domains. Half of the number of ABC transporters are comprised of one TMD (transmembrane domain) and one NBD (nucleotide-binding domain). Transporters also exist that do not contain a TMD domain, and instead consist of two NBD domains. The NBD domain is present in all structural types and contains characteristic motifs, including the D loop, which is responsible for keeping dimers together, the H switch loop, which interacts with the transmembrane domain, the Walker A and Walker B motifs, which form the P loop and is associated with ATP, and finally the characteristic Q and H loop, as well as the signature motif, which only occurs in ABC proteins, thereby making it possible to distinguish them from other ATPases. ABC transporters can be classified according to the TMD domain presence, function, protein solubility, and amino acid sequence. One classification is the division of transporters into eight subfamilies , of which ABCH subfamilies have not been identified in plants. The ABCE and ABCF subfamilies belong to soluble proteins devoid of the TMD domain. One study has shown these transporters to be important in sequestration, and they impart herbicide resistance, since they can sequester glyphosate in vacuoles ,85.In order for a plant to properly develop under stress conditions, such as the presence of heavy metals, numerous rhizospheric microorganisms are needed to stimulate plant development. Known rhizospheric microorganisms are the symbiotic papillary bacteria of legumes, bacteria, and fungi, which stimulate plant growth and mycorrhizal fungi. These microorganisms can release substances into the rhizosphere that change the soil physical and chemical nature, thus modifying soil metal bioavailability .P. sylvestris, form ectomycorrhizae with Scleroderma citrinum, Lactarius rufus, Rhizopogon roseolus, and Amanita muscaria. They accumulate heavy metals in root or rhizomorph muffs, which contribute to the nontransportation of metals to above-ground plant sections. Ectomycorrhizal fungi secrete exudates to facilitate metal ion bonding in muffs, and to limit metal ion penetration into the root cells, by attaching them to polysaccharides in the fungal hyphae cell walls. Approximately 120 fungal species from the order Glomales participate in arbuscular mycorrhizae, primarily increasing the soil nutrient bioavailability. Endomycorrhizae occur in herbaceous plants as well as in ash, maple, and fruit trees [Fungi form a symbiotic relationship with the plant root system (mycorrhizae). Mycelia that develop on the root surface are known as external mycorrhizae (ectomycorrhizae), while mycelia that develop inside the root are known as arbuscular mycorrhizae (endomycorrhizae). A characteristic structure found in ectomycorrhizae are the muffs produced by the mycelium. The mycelium penetrates the root cortex cells and create a Hartig net, i.e., a channel for substance exchange between the fungus and plant. Other characteristic elements are rhizomorphs and loose hyphae, which, together with muffs, store and exchange mineral salts and water. Trees characterized by increased heavy metal concentrations, e.g., it trees .Thelephora terrestris, increases plant zinc levels, while Glomus caledonium increases the Cu2+ concentration in maize, but only when these metals have a low soil level. The fungi limit their uptake when these metals reach high concentrations. Arbuscular mycorrhizal fungi also play a protective role, an example of which is Glomus intraradices, and reduce toxic cadmium effects in P. sativum. Endomycorrhizae also reduce stress caused by water scarcity: the fungi produce more metal-binding substances and improve nutrition with mineral salts. Plants in symbiosis with fungi also contain elevated root phenol levels, which, in the presence of high cadmium concentrations, causes cell wall stiffening, which makes the penetration of aqueous Cd2+ solutions more difficult [Mycorrhizal fungi significantly increase heavy metal plant resistance, but these defense mechanisms may vary depending on the fungus, the metal type, and its concentration. The pine tree ectomycorrhizal fungus, ifficult ,88.Actinobacteria, Streptomyces, and Pseudomonas [Bacteria stimulating plant growth and development, i.e., PGPR (plant growth-promoting rhizobacteria), increase plant heavy metal tolerance. They affect metal mobility and availability through the release of chelating agents, acidification, phosphate solubilization, and changes in the rate of metal oxidation. Examples of PGPR bacteria that improve plant growth in areas with high zinc and lead levels are udomonas .3+ in the soil, are released through the cell membrane anion channel and secreted into the rhizosphere. Where the aluminum ions are most concentrated, the metal is bound and immobilized. In addition to organic acids, plants can also secrete phenolic compounds  into the rhizosphere, thereby improving the Al3+ immobilization mechanism, but only if the plant has first been treated with silicon [Metal chelators are substances secreted by plants into the soil that form insoluble metal complexes, i.e., they immobilize them while simultaneously protecting the plant against toxic substance uptake. Such substances are organic acid anions , which, when activated by the presence of Al silicon . When th silicon .Other mechanisms that limit heavy metal uptake, i.e., stress factor avoidance mechanisms, are increased pH, which contributes to decreased metal uptake, and the formation of a root oxidizing zone, which involves metal oxidizing, thereby reducing their availability .The cell wall is the first barrier separating the plant from the external environment and is therefore important in protecting the plant against heavy metal penetration. Cell wall metal accumulation contributes to increased plant tolerance to metals and partakes in detoxification. In studies on different soybean varieties with high cadmium concentration tolerance and metal sensitivity, differences in cell wall composition were found. It was shown that 48.5–75.5% of Cd had accumulated in the cell wall. Pectin, hemicellulose, and cellulose are the main cell wall components and are mostly polysaccharides, which, apart from monosaccharides, may contain uronic acids. Pectin and hemicellulose, as main components, contain uronic acids, which bind to heavy metals .3+, Cd2+, and Cu2+ binding, and can be determined by pectin methylesterase activity, which transforms pectin into a loose carboxylic structure with the release of methanol and protons [The binding of metals in a cell wall may depend not only on differences in its composition, but also on the chemical form of pectin, which in turn depends on the degree of esterification. A high degree of demethylated esterification contributes to increased Al protons ,91.A. thaliana, a plant that is sensitive to Cd stress, while in N. caerulescens, the expression of these genes remains at a high and constant level [Moreover, under the influence of high Cd levels, lignin in the secondary cell wall causes wall hardening, i.e., its lignification, which protects the plant from being crushed and facilitates water and mineral salt conduction. Lignin is an important cell wall component because it forms complexes with metals such as Cd, Cu, Zn, as well as cellulose, and the number of these complexes affects cell wall permeability, thus affecting heavy metal tolerance and uptake . Sensitint level .Another mechanism that is also an indicator of plant stress is the production of components that create a barrier against metal penetration into the plant, such as callose, cutin, and lignin. If Al, Co, Ni, Zn, and Pb concentrations are high, the plant synthesizes callose on the outer cell membrane surface within a few minutes, thereby producing a layer which is impermeable to metals. Moreover, all these modifications cause the primary cell wall to assume the character of a secondary wall due to changes such as an increased number of transverse bonds between polysaccharide molecules, lignin production, phenolic substances, and a greater cell wall thickness. These changes contribute to the formation of a structure which is flexible and permeable to aqueous solutions, and is compact, rigid, and thick, and consequently impermeable to metals .Heavy metal tolerance mechanisms are activated when metals penetrate the cells and where their presence is most dangerous for the plant. One of these mechanisms consists of reducing the activity of cell membrane transporters that uptake metal ions from the rhizosphere, which is in turn caused by the increased expression of genes encoding the transporters, located in the tonoplasm, and causing metal storage in the vacuole .Metals such as Hg, Pb, or Cd can interact with proteins and DNA, and can damage nucleic acids and inhibit protein folding. The inhibition of protein folding under stress conditions leads to protein inactivity, thereby making them nonfunctional. Therefore, plant cells activate proteins, i.e., chaperones, which can repair these proteins and protect them from developing when heavy metal are in excess .2+-binding and transportation in A. thaliana is similar to that found in yeast. In A. thaliana, copper is absorbed into the cell cytoplasm by COPT membrane transporters, where it is bound to the chaperone CCH (copper chaperone). The copper complex from CCH is transported to Golgi apparatus vesicles. Hereafter, Cu2+ passes to the follicular lumen via transporters, and is incorporated into the ERT1 (ethanol regulator of translation 1) protein, which is transported and built into the cell membrane where it acts as an ethylene receptor. The mechanism by which copper ions are delivered to the mitochondria, with the help of the AtCOX17 (cytochrome C oxidase copper chaperone) chaperone, is also known. An increased activity of these proteins has been observed when copper ions are excessive, as well as bacterial infections and NO or H2O2, which disturb mitochondrial functioning [Metals that are toxic or in excess within the cell can be bound by chaperone proteins. These proteins then transport heavy metal ions to where they can be built into specific molecules or structures and perform specific functions . The tolctioning .To prevent toxic heavy metal effects in the cytoses, plant cells have developed a number of detoxification mechanisms, of which metal ion chelation is the main mechanism. Chelation involves the binding of metal ions by cysteine-rich peptides to form a nontoxic complex, which is sequestered in a vacuole to prevent the free movement of metals in the cytosol. The most important classes of cysteine-rich peptides that are involved in chelation are the synthesized enzymes phytochelatins (PC), known as class III metallothioneins (MT), as well as class I and II metallothioneins, which are primary gene products. The heavy metals found in the cytosol can also be bound by glutathione, organic acids, nicotianamine, and amino acids .A. thaliana, belonging to four forms classified by the type of cysteine residue. These are MT1a, MT2a, MT2b, MT3, MT4a, and MT4b. Most of these MTs contribute to Cu tolerance; however, MT1, 2, and 3 are also able to bind Cd, and MT4 can bind and accumulate Zn. In cases of high Cd concentrations in A. thaliana, MT1a binds and accumulates cadmium, thereby increasing the plant’s tolerance to the metal. N. caerulescens has shown a higher level of MT1 and MT2 compared to A. thaliana, which makes it more tolerant to Cd and able to store higher Cd concentrations [Metallothionein (MT) is a cysteine-rich protein with a low molecular weight and is able to bind metal ions. Six MTs have been identified in trations ,94.2+ ions, which stimulate PC synthesis by up to six times as much as Cu2+ or Zn2+ ions. Additionally, phytochelatins are the first to be synthesized and accumulated within the roots. Moreover, phytochelatins increasingly accumulated in S. nigrum L. treated with high Cu concentrations, which contributed to binding and immobilization of excess copper, making its transport to the shoots impossible [Phytochelatins are low-molecular-weight peptides consisting of glutamic acid, cysteine, and glycine. These peptides are synthesized from glutathione (GSH) by phytochelatin synthase (PCS), an enzyme which requires post-translational modification to be active ,98. Phytpossible . In the possible .A. thaliana treated with cadmium, which increases its tolerance to this metal, while a reduction in GSH levels contributed to a decreased Cd tolerance. Rice studies have found that plants which can tolerate Cd have higher glutathione levels than plants sensitive to this metal. When the cytoplasmic cadmium concentration is high, a GSH–metal complex forms, which has a higher affinity for phytochelatin synthase than free heavy metal ions, and which causes its activation. Activation is achieved by protein folding and conformational changes, resulting in phytochelatin synthesis. GSH plays an important role as a substrate in phytochelatin synthesis [Glutathione is a tripeptide containing a thiol group, which makes it capable of capturing metals, and it is also a cellular antioxidant and signal molecule for reactive oxygen species (ROS). An increased expression of genes responsible for glutathione synthesis has been observed in ynthesis ,97.Nicotianamine (NA) is a nonprotein amino acid which binds metals such as Zn, Ni, Co, Mn, and Fe. NA is synthesized by the enzyme nicotianamine synthase (NAS), which has a different number of genes in different species. The expression of these genes occurs when heavy metals are in excess, and are responsible for increased NA concentrations; thus, they help to maintain metal homeostasis. Only one NAS gene is known from Solanaceae plants, while the Poaceae has three genes, and the Brassicaceae has four, namely: NAS1, 2, 3, and 4 .A. thaliana when zinc is in excess, which results in increased Zn transport to root cell vacuoles, where a NA–Zn complex is formed, thereby making its transport to the shoots impossible, where zinc is more toxic [Metal–NA complexes can be transported through the cell membrane due to membrane transporters from the YSL family. In tonoplasts, the ZIF1 (zinc-induced facilitator (1)) transporter is responsible for transporting NA from the cytoplasm to the vacuole, where the metal–NA complex is formed. The ZIF1 gene is expressed in re toxic .Heat-shock proteins (HSP) are characterized by a conservative primary structure, and are produced in response to abiotic stress, such as high temperature, oxidative stress, UV radiation, and high heavy metal concentrations. HSP proteins can be categorized into five classes: the HSP60 family (chaperonins), the HSP70/DnaK family, the HSP90 family, the HSP100/ClpB family, and the small heat shock proteins (sHSP) family ,102,103.Arabidopsis [In rice, 25 HSP proteins have been identified, while 38 have been identified in soybean and 21 in bidopsis . Under tbidopsis ,101. Morbidopsis .O. sativa, S. oleracea L., and A. thaliana, with 18 identified in A. thaliana and 32 in O. sativa. A total of 61 HSP70 genes were identified during the analysis of HSP proteins in N. tabacum [The best known and most conservative is the HSP70 protein family. It is constructed from three domains, namely: the N-terminal domain, interdomain, and C-terminal domain. The C-terminal domain binds to the substrate, while the N-terminal domain binds ATP. Genes encoding HSP70 proteins have been identified in many plant species, including  tabacum .Genetic control is one of the mechanisms for the development of plant heavy metal tolerance. Heavy metal tolerance can be associated with the overexpression of associated genes or with the repression of these genes. There are three models of genetic control mechanisms associated with heavy metal tolerance. The first model consists of one main gene, which can interact with other genes capable of changing the expression of the main gene. The next model is a multigenous model, where there are a small number of genes, with each gene having an equal effect on tolerance. The polygenic model is a mechanism where metal tolerance is associated with a different number of genes, and each of them has a small influence on the tolerance variation .Recent studies have shown a strong influence of microRNA (miRNA) in the modulation of gene expression. This is a nonprotein RNA constructed from about 21 nucleotides, which performs the role of gene expression modulation at the post-transcriptional level. The miRNAs are involved in the abiotic stress response. Under stress conditions, plants also activate the mechanism of miRNA-mediated post-transcriptional regulation. Rice studies showed changes in the expression of 19 miRNAs caused by excessive Cd levels. However, a high expression level was only observed for miR528, and the level of the remaining miRNAs was reduced ,102.Ricinus communis L. in conditions of metal toxicity demonstrated miRNA level changes, which translate into expression level changes of the target genes. The levels of seven miRNAs involved in the stress response caused by heavy metals have been studied. It was shown that, under elevated Ni2+ conditions, the level of miR398 was reduced and the target gene overexpressed. It is also known that most plant miRNAs are regulated by transcription factors (TF) [Mature miRNAs attached to the RISC (RNA-induced silencing complex) cause the target mRNA expression to be downregulated by chopping up, degrading, or blocking translation. Studies on ors (TF) ,102.T. caerulescens and WRKY4 in Z. mays [Transcription factors act as regulators in response to stress by regulating the expression of genes associated with stress factor tolerance. Under toxic cadmium conditions, TF regulates the expression of target genes such as CaPF1 and HsfA4a. Studies on TFs of the WRKY family have shown that the transcription of WRKY12 is inhibited in the presence of high Cd levels. The WRKY family is involved in the stress responses of many plants, e.g., in WRKY53 in  Z. mays .B. nigra, 88 genes with a different level of mRNA in the roots and 24 genes in the shoots were identified in response to high Cd levels. These genes were associated with metal accumulation, chelation, and transport [Many genes are involved in heavy metal stress responses. In ransport . In the ransport .Thanks to the increasingly rapid development of molecular techniques, it is possible to perform more accurate heavy metal hyperaccumulator analyses. They allow the discovery of the unknown mechanisms that induce plants to become hyperaccumulators, thus creating more opportunities for more efficient phytoremediation of contaminated soils see .