Lanternarene-Based Self-Sorting Double-Network Hydrogels for Flexible Strain Sensors.

Conductive flexible hydrogels have attracted immense attentions recently due to their wide applications in wearable sensors. However, the poor mechanical properties of most conductive polymer limit their utilizations. Herein, a double network hydrogel is fabricated via a self-sorting process with cationic polyacrylamide as the first flexible network and the lantern[33]arene-based hydrogen organic framework nanofibers as the second rigid network. This hydrogel is endowed with good conductivity (0.25 S m-1) and mechanical properties, such as large Young's modulus (31.9 MPa), fracture elongation (487%) and toughness (6.97 MJ m-3). The stretchability of this hydrogel is greatly improved after the kirigami cutting, which makes it can be used as flexible strain sensor for monitoring human motions, such as bending of fingers, wrist and elbows. This study not only provides a valuable strategy for the construction of double network hydrogels by lanternarene, but also expands the application of the macrocycle hydrogels to flexible electronics.

Mixed probiotics modulated gut microbiota to improve spermatogenesis in bisphenol A-exposed male mice.

Bisphenol A (BPA), an environmental endocrine disruptor (EDC), has been implicated in impairing intestinal and male reproductive dysfunction. The efficacy of gut microbiota modulation for BPA-exposed testicular dysfunction has yet to be verified through research. Therefore, this study explored the potential of mixed probiotics in restoring spermatogenesis damage through the gut-testis axis under BPA exposure. We selected two probiotics strains (Lactobacillus rhamnosus and Lactobacillus plantarum) with BPA removal properties in vitro and the BPA-exposed male mice model was established. The probiotics mixture effectively reduced BPA residue in the gut, serum, and testis in mice. Through 16 S rDNA-seq and metabolomics sequencing, we uncovered that vitamin D metabolism and bile acid levels in the gut was abolished under BPA exposure. This perturbation was linked to an increased abundance of Faecalibaculum and decreased abundance of Lachnospiraceae_NK4A136_group and Ligilactobacillus. The probiotics mixture restored this balance, enhancing intestinal barrier function and reducing oxidative stress. This improvement was accompanied by a restored balance of short-chain fatty acids (SCFAs). Remarkably, the probiotics ameliorated testicular dysfunction by repairing structures of seminiferous tubules and reversing arrested spermiogenesis. Further, the probiotics mixture enhanced testosterone-driven increases in spermatogonial stem cells and all stages of sperm cells. Testicular transcriptome profiling linked these improvements to fatty acid degradation and peroxisome pathways. These findings suggest a significant interplay between spermatogenesis and gut microbiota, demonstrating that probiotic intake could be a viable strategy for combating male subfertility issues caused by BPA exposure.

A Review of the Use of Native and Engineered Probiotics for Colorectal Cancer Therapy.

Colorectal cancer (CRC) is a serious global health concern, and researchers have been investigating different strategies to prevent, treat, or support conventional therapies for CRC. This review article comprehensively covers CRC therapy involving wild-type bacteria, including probiotics and oncolytic bacteria as well as genetically modified bacteria. Given the close relationship between CRC and the gut microbiota, it is crucial to compile and present a comprehensive overview of bacterial therapies used in the context of colorectal cancer. It is evident that the use of native and engineered probiotics for colorectal cancer therapy necessitates research focused on enhancing the therapeutic properties of probiotic strains.. Genetically engineered probiotics might be designed to produce particular molecules or to target cancer cells more effectively and cure CRC patients.

High altitude Relieves transmission risks of COVID-19 through meteorological and environmental factors: Evidence from China.

Existing studies reported higher altitudes reduce the COVID-19 infection rate in the United States, Colombia, and Peru. However, the underlying reasons for this phenomenon remain unclear. In this study, regression analysis and mediating effect model were used in a combination to explore the altitudes relation with the pattern of transmission under their correlation factors. The preliminary linear regression analysis indicated a negative correlation between altitudes and COVID-19 infection in China. In contrast to environmental factors from low-altitude regions (<1500 m), high-altitude regions (>1500 m) exhibited lower PM2.5, average temperature (AT), and mobility, accompanied by high SO2 and absolute humidity (AH). Non-linear regression analysis further revealed that COVID-19 confirmed cases had a positive correlation with mobility, AH, and AT, whereas negatively correlated with SO2, CO, and DTR. Subsequent mediating effect model with altitude-correlated factors, such as mobility, AT, AH, DTR and SO2, suffice to discriminate the COVID-19 infection rate between low- and high-altitude regions. The mentioned evidence advance our understanding of the altitude-mediated COVID-19 transmission mechanism.

Feed-additive Limosilactobacillus fermentum GR-3 reduces arsenic accumulation in Procambarus clarkii.

Procambarus clarkii (crayfish) accumulates a high concentration of Arsenic (As) from the aquatic environment and causes considerable human health risks. In this study, Limosilactobacillus fermentum GR-3 strain was isolated from "Jiangshui" and applied for As(III) adsorption and antioxidant abilities. Strain GR-3 removed 50.67% of 50 mg/L As(III) and exhibited the high antioxidant potential of DPPH (1,1-Diphenyl-2-picrylhydrazyl) (87.63%) and hydroxyl radical (74.51%) scavenging rate in vitro. P. clarkii was feed with strain GR-3, the results showed that As(III) concentration reduced, and residual level in hepatopancreas was decreased by 36%, compared to As(III)-exposed group (control). Gut microbial sequencing showed that strain GR-3 restores gut microbiota dysbiosis caused by As(III) exposure. Further application in the field scale was performed and revealed a decrease in As(III) accumulation and increasing 50% aquaculture production of the total output. In summary, feed-additive probiotic is recommended as a novel strategy to minimize aquaculture foods toxicity and safe human health.

Cu(II) nonspecifically binding chromate reductase NfoR promotes Cr(VI) reduction.

Cu(II)-enhanced microbial Cr(VI) reduction is common in the environment, yet its mechanism is unknown. The specific activity of chromate reductase, NfoR, from Staphylococcus aureus sp. LZ-01 was augmented 1.5-fold by Cu(II). Isothermal titration calorimetry and spectral data show that Cu(II) binds to NfoR nonspecifically. Further, Cu(II) stimulates the nitrobenzene reduction of NfoR, indicating that Cu(II) promotes electron transfer. The crystal structure of NfoR in complex with CuSO4 (1.46 Å) was determined. The overall structure of NfoR-Cu(II) complex is a dimer that covalently binds with FMN and Cu(II)-binding pocket is located at the interface of the NfoR dimer. Structural superposition revealed that NfoR resembles the structure of class II chromate reductase. Site-directed mutagenesis revealed that Leu46 and Phe123 were involved in NADH binding, whereas Trp70 and Ser45 were the key residues for nitrobenzene binding. Furthermore, His100 and Asp171 were preferential affinity sites for Cu(II) and that Cys163 is an active site for FMN binding. Attenuation reductase activity in C163S can be partially restored to 54% wild type by increasing Cu(II) concentration. Partial restoration indicates dual-channel electron transfer of NfoR via Cu(II) and FMN. We propose a catalytic mechanism for Cu(II)-enhanced NfoR activity in which Cu(I) is formed transiently. Together, the current results provide an insight on Cu (II)-induced enhancement and benefit of Cr(VI) bioremediation.

A copper-specific microbial fuel cell biosensor based on riboflavin biosynthesis of engineered Escherichia coli.

Copper pollution poses a serious threat to the aquatic environment; however, in situ analytical methods for copper monitoring are still scarce. In the current study, Escherichia coli Rosetta was genetically modified to express OprF and ribB with promoter Pt7 and PcusC , respectively, which could synthesize porin and senses Cu2+ to produce riboflavin. The cell membrane permeability of this engineered strain was increased and its riboflavin production (1.45-3.56 µM) was positively correlated to Cu2+ (0-0.5 mM). The biosynthetic strain was then employed in microbial fuel cell (MFC) based biosensor. Under optimal operating parameters of pH 7.1 and 37°C, the maximum voltage (248, 295, 333, 352, and 407 mV) of the constructed MFC biosensor showed a linear correlation with Cu2+ concentration (0.1, 0.2, 0.3, 0.4, 0.5 mM, respectively; R2 = 0.977). The continuous mode testing demonstrated that the MFC biosensor specifically senses Cu2+ with calculated detection limit of 28 µM, which conforms to the common Cu2+ safety standard (32 µM). The results obtained with the developed biosensor system were consistent with the existing analytical methods such as colorimetry, flame atomic absorption spectrometry, and inductively coupled plasma optical emission spectrometry. In conclusion, this MFC-based biosensor overcomes the signal conversion and transmission problems of conventional approaches, providing a fast and economic analytical alternative for in situ monitoring of Cu2+ in water.

Metatranscriptomic analysis reveals active microbes and genes responded to short-term Cr(VI) stress.

Heavy metals have been severely polluting the environment. However, the response mechanism of microbial communities to short-term heavy metals stress remains unclear. In this study, metagenomics (MG) and metatranscriptomics (MT) was performed to observe the microbial response to short-term Cr(VI) stress. MG data showed that 99.1% of species were similar in the control and Cr(VI) treated groups. However, MT data demonstrated that 83% of the microbes were active in which 58.7% increased, while the relative abundance of 41.3% decreased after short-term Cr(VI) incubation. The MT results also revealed 9% of microbes were dormant in samples. Genes associated with oxidative stress, Cr(VI) transport, resistance, and reduction, as well as genes with unknown functions were 2-10 times upregulated after Cr(VI) treatment. To further confirm the function of unknown genes, two genes (314 and 494) were selected to detect the Cr(VI) resistance and reduction ability. The results showed that these genes significantly increased the Cr(VI) remediation ability of Escherichia coli. MT results also revealed an increase in the expression of some rare genera (at least two times) after Cr(VI) treatment, indicating these rare species played a crucial role in microbial response to short-term Cr(VI) stress. In summary, MT is an efficient way to understand the role of active and dormant microbes in specific environmental conditions.

Mechanisms and Advances in Anti-Ovarian Cancer with Natural Plants Component.

Ovarian cancer ranks seventh in the most common malignant tumors among female disease, which seriously threatens female reproductive health. It is characterized by hidden pathogenesis, missed diagnosis, high reoccurrence rate, and poor prognosis. In clinic, the first-line treatment prioritized debulking surgery with paclitaxel-based chemotherapy. The harsh truth is that female patients are prone to relapse due to the dissemination of tumor cells and drug resistance. In these circumstances, the development of new therapy strategies combined with traditional approaches is conductive to improving the quality of treatment. Among numerous drug resources, botanical compounds have unique advantages due to their potentials in multitarget functions, long application history, and wide availability. Previous studies have revealed the therapeutic effects of bioactive plant components in ovarian cancer. These natural ingredients act as part of the initial treatment or an auxiliary option for maintenance therapy, further reducing the tumor and metastatic burden. In this review, we summarized the functions and mechanisms of natural botanical components applied in human ovarian cancer. We focused on the molecular mechanisms of cell apoptosis, autophagy, RNA and DNA lesion, ROS damage, and the multiple-drug resistance. We aim to provide a theoretical reference for in-depth drug research so as to manage ovarian cancer better in clinic.

Microbial Fuels Cell-Based Biosensor for Toxicity Detection: A Review.

With the unprecedented deterioration of environmental quality, rapid recognition of toxic compounds is paramount for performing in situ real-time monitoring. Although several analytical techniques based on electrochemistry or biosensors have been developed for the detection of toxic compounds, most of them are time-consuming, inaccurate, or cumbersome for practical applications. More recently, microbial fuel cell (MFC)-based biosensors have drawn increasing interest due to their sustainability and cost-effectiveness, with applications ranging from the monitoring of anaerobic digestion process parameters (VFA) to water quality detection (e.g., COD, BOD). When a MFC runs under correct conditions, the voltage generated is correlated with the amount of a given substrate. Based on this linear relationship, several studies have demonstrated that MFC-based biosensors could detect heavy metals such as copper, chromium, or zinc, as well as organic compounds, including p-nitrophenol (PNP), formaldehyde and levofloxacin. Both bacterial consortia and single strains can be used to develop MFC-based biosensors. Biosensors with single strains show several advantages over systems integrating bacterial consortia, such as selectivity and stability. One of the limitations of such sensors is that the detection range usually exceeds the actual pollution level. Therefore, improving their sensitivity is the most important for widespread application. Nonetheless, MFC-based biosensors represent a promising approach towards single pollutant detection.

Enhancing tumor-specific recognition of programmable synthetic bacterial consortium for precision therapy of colorectal cancer.

Probiotics hold promise as a potential therapy for colorectal cancer (CRC), but encounter obstacles related to tumor specificity, drug penetration, and dosage adjustability. In this study, genetic circuits based on the E. coli Nissle 1917 (EcN) chassis were developed to sense indicators of tumor microenvironment and control the expression of therapeutic payloads. Integration of XOR gate amplify gene switch into EcN biosensors resulted in a 1.8-2.3-fold increase in signal output, as confirmed by mathematical model fitting. Co-culturing programmable EcNs with CRC cells demonstrated a significant reduction in cellular viability ranging from 30% to 50%. This approach was further validated in a mouse subcutaneous tumor model, revealing 47%-52% inhibition of tumor growth upon administration of therapeutic strains. Additionally, in a mouse tumorigenesis model induced by AOM and DSS, the use of synthetic bacterial consortium (SynCon) equipped with multiple sensing modules led to approximately 1.2-fold increased colon length and 2.4-fold decreased polyp count. Gut microbiota analysis suggested that SynCon maintained the abundance of butyrate-producing bacteria Lactobacillaceae NK4A136, whereas reducing the level of gut inflammation-related bacteria Bacteroides. Taken together, engineered EcNs confer the advantage of specific recognition of CRC, while SynCon serves to augment the synergistic effect of this approach.

Tumor-targeting engineered probiotic Escherichia coli Nissle 1917 inhibits colorectal tumorigenesis and modulates gut microbiota homeostasis in mice.

AIMS: Preliminary studies have identified the use of probiotics as a potential treatment strategy against colorectal cancer (CRC). However, natural probiotics lack direct tumor-targeting and tumor-killing activity in the intestine. This study aimed to construct a tumor-targeting engineered probiotic to combat CRC. MAIN METHODS: Standard adhesion assay was performed to analyze the adherence ability of tumor-binding protein HlpA to CT26 cells. CCK-8 assay, Hoechst 33258 staining and flow cytometry analysis were used for examining cytotoxicity of tumoricidal protein azurin toward CT26 cells. An engineered probiotic Ep-AH harboring azurin and hlpA genes was developed using Escherichia coli Nissle 1917 (EcN) chassis. Antitumor effects of Ep-AH were evaluated in the azoxymethane (AOM) and dextran sodium sulfate salt (DSS)-induced CRC mice. Moreover, analysis of gut microbiota was conducted via fecal 16S rRNA gene sequencing and shotgun metagenomic sequencing. KEY FINDINGS: Azurin caused a dose-dependent increase of apoptosis in CT26 cells. Ep-AH treatment reversed weight loss (p < 0.001), fecal occult blood (p < 0.01), and shortening of colon length (p < 0.001) than model group, as well as reducing tumorigenesis by 36 % (p < 0.001). Both Ep-H and Ep-A (EcN expressing HlpA or azurin) were less effective than Ep-AH. Furthermore, Ep-AH enriched the members of beneficial bacteria (e.g., Blautia and Bifidobacterium) and reversed abnormal changes of genes associated with several metabolic pathways (e.g., lipopolysaccharide biosynthesis). SIGNIFICANCE: These results demonstrated that Ep-AH had excellent therapeutic benefits on cancer remission and gut microbiota modulation. Our study provides an effective strategy for anti-CRC treatment.

NahAa can convert naphthalene and reduce chromate simultaneously and immobilized on functional multiwall carbon nanotubes for wastewater treatment.

Pseudomonas brassicacearum LZ-4 is a facultative anaerobic bacterium, can efficiently degrade naphthalene and reduce chromate simultaneously. In this study, we showed that the naphthalene degradation enzyme NahAa from P. brassicacearum LZ-4 can reduce Cr(VI). Heterologous expression in E. coli S17-1 along with RNA interference of NahAa in strain LZ-4 showed the enzyme can reduce chromate in vivo. In vitro, purified NahAa was identified and can catalyze Cr(VI) reduction by 64.2%. Flavin adenine dinucleotide (FAD) was identified as a cofactor of NahAa, which Cr(VI) could obtain electrons from NADH through NahAa-associated FAD for reduction. Immobilized NahAa on functional multi walled carbon nanotubes via physical adsorption method to produce a stable, high efficient composite MWCNT-NahAa. The maximum efficiency of MWCNT-NahAa composite was obtained in enzyme concentrations of 6 mg/mL and 20 min immobilization time. The optical reaction conditions for MWCNT-NahAa were pH 7.0 and 30 °C, still retaining 50% of its initial activities after five consecutive cycles. Application of composites in wastewater can reduce 90.4% Cr(VI), higher than free NahAa that was 63.5%. To our best knowledge, this is the first report immobilized enzyme in polycyclic aromatic hydrocarbons-degradation pathway for Cr(VI) wastewater treatment, providing a new insights on combined pollution remediation.

Chitooligosaccharides production from shrimp chaff in chitosanase cell surface display system.

Chitin refers to a natural biopolymer, which is economically significant to next-generation biorefineries. In this study, a novel high-yield method with cell surface-display chitosanase (CHI-1) was built to produce chitooligosaccharides (COS) from shrimp chaff through the co-fermentation in the presence of Bacillus subtilis and Acetobacter sp. Under the optimized co-fermentation conditions (5 g/L yeast extracts, 10 g/L KH2PO4, 6% ethanol, 50 g/L glucose), the final deproteinization (DP) and demineralization (DM) efficiency and the chitin yield were achieved as 94, 92 and 18%, respectively. The engineered E. coli BL21-pET23b(+)-NICHI maintained 81% of the initial enzyme activity after 40 days at room temperature. The crude CHI-1 was inactivated after one-day interacting with prepared chitosan. Moreover, E. coli BL21-pET23b(+)-NICHI still maintained excellent hydrolysis ability in 7 days, and the COS yield reached 41%. Accordingly, the proposed method exhibited excellent stability and a high hydrolysis efficiency to produce COS with whole engineered cells.

SARS-CoV-2 triggered oxidative stress and abnormal energy metabolism in gut microbiota.

Specific roles of gut microbes in COVID-19 progression are critical. However, the circumstantial mechanism remains elusive. In this study, shotgun metagenomic or metatranscriptomic sequencing was performed on fecal samples collected from 13 COVID-19 patients and controls. We analyzed the structure of gut microbiota, identified the characteristic bacteria, and selected biomarkers. Further, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were employed to correlate the taxon alterations and corresponding functions. The gut microbiota of COVID-19 patients was characterized by the enrichment of opportunistic pathogens and depletion of commensals. The abundance of Bacteroides spp. displayed an inverse relationship with COVID-19 severity, whereas Actinomyces oris, Escherichia coli, and Streptococcus parasanguini were positively correlated with disease severity. The genes encoding oxidoreductase were significantly enriched in gut microbiome of COVID-19 group. KEGG annotation indicated that the expression of ABC transporter was upregulated, while the synthesis pathway of butyrate was aberrantly reduced. Furthermore, increased metabolism of lipopolysaccharide, polyketide sugar, sphingolipids, and neutral amino acids were found. These results suggested the gut microbiome of COVID-19 patients was in a state of oxidative stress. Healthy gut microbiota may enhance antiviral defenses via butyrate metabolism, whereas the accumulation of opportunistic and inflammatory bacteria may exacerbate COVID-19 progression.

Enhanced removal of trivalent chromium from leather wastewater using engineered bacteria immobilized on magnetic pellets.

Leather wastewater contains various toxic contaminants, with trivalent chromium (Cr(III)) having high concentration and adversely affecting wastewater treatment. In this study, a Cr(III) adsorption protein (MerP) was displayed on the cell surface of Escherichia coli and then coupled with a magnetic pellet system to facilitate Cr(III) adsorption. The results showed the engineered strain M-BL21 achieved an in vitro Cr(III) adsorption capacity of 2.38 mmol/g. Next, the magnetic pellets were prepared as component ratios of sodium alginate (2.5%), polyvinyl alcohol (8%), Fe3O4 nanoparticles (3.5%), and M-BL21 at 3 g/L. The optimized system was capable of Cr(III) adsorption at an efficiency of 91.29%, which was substantially higher than that of the magnetic carrier alone (67%). Results of scanning electron microscopy with energy-dispersive X-ray analysis proved that Cr(III) was absorbed on the magnetic pellet. The recyclable performance of magnetic property (13.34185 emu/g) and high Cr(III) adsorption efficiency (68.75%) remained after five cycles of Cr(III) absorption. In the medium-scale experiment, 25 L of leather wastewater were treated with magnetic pellet and the Cr(III) removal efficiency reached 88.2%. Thus, our results present an advanced, fully operational, and eco-friendly method for in situ removal of Cr(III) from contaminated wastewater.

Bioaugmentation improves the anaerobic co-digestion of cadmium-containing plant residues and cow manure.

Phytoremediation causes a large quantity of phytoremediation residues rich in heavy metals (HMs). This kind of plant residue can be used as a substrate for anaerobic digestion (AD) to reduce the content of HM-containing biomass, but high concentrations of HMs will inhibit the digestion efficiency and reduce the conversion efficiency of plant residues. Bioaugmentation may be an effective method to improve the degradation efficiency and methane yield of plant residues rich in HMs. In this study, a cellulose-degrading anaerobic bacteria Paracoccus sp. Termed strain LZ-G1 was isolated from cow dung, which can degrade cellulose and simultaneously adsorb Cd2+. The Cd2+ (10 mg/L)-adsorbtion efficiency and cellulose (463.12 g/kg)-degradation rate were 65.1 % and 60.59 %, respectively. In addition, using the strain LZ-G1 bioaugmented Cd2+-containing plant residues and cow manure mixed AD system, the system's biogas and methane production significantly increased (98.97 % and 142.03 %, respectively). During the AD process, the strain LZ-G1 was successfully colonized in the digestion system. Furthermore, the microbial community analysis revealed that LZ-G1 bioaugmentation alleviates the toxicity of free Cd2+ to the microbial community in the AD system, regulates and restores the archaea genus dominant in the methanogenesis stage, and restores the relative abundance of dominant bacteria associated with biomass hydrolysis. The restoration of the microbial community increased the biogas yield and methane production rate. Thus, bioaugmentation provides an easy and a feasible method for the actual on-site treatment of HM-rich phytoremediation residues.

Gut Escherichia coli expressing Pb2+-adsorption protein reduces lead accumulation in grass carp, Ctenopharyngodon idellus.

Lead (Pb2+) is easy to accumulate in fish which become a major source of Pb2+ exposure to humans. In this study, a recombinant Escherichia coli strain expressing Pb2+-specific surface-binding protein anchored by the ice nucleation protein was introduced into grass carp (Ctenopharyngodon idellus) gut to investigate its protective effect against dietary Pb2+ exposure. Pb2+ mostly precipitated on the surface of the engineered strain through Pb2+-specific surface-binding protein, with a maximum adsorption efficiency of 73% and an adsorption capacity of 163 µmol/g dry cells. The Pb2+ concentration in engineered bacteria-fed grass carp was reduced significantly, and the residual level of Pb2+ in feces was increased by 76%, compared with the control group. Meantime, the engineered bacteria were able to mitigate the oxidative stress and histological alterations of intestines and dysbiosis of gut microbiota induced by Pb2+exposure. Thus, the engineered bacterium that can effectively reduce Pb2+ residue in grass carp might be a useful tool for decontamintion of lead in aquatic organisms.

Feed-additive of bioengineering strain with surface-displayed laccase degrades sulfadiazine in broiler manure and maintains intestinal flora structure.

Sulfonamide antibiotics (SAs) are excreted into the ecosystem unchanged through feces and urine because of their low adsorption and degradation in the guts of humans and animals. In this study, a novel whole-cell biocatalyst with fungal laccase on the cell surface of Escherichia coli Nissle 1917 was developed to degrade sulfadiazine (SDZ). Engineered strain EcN-IL showed laccase enzyme activity of 2 ± 1 U/mg dry weight of cell and degraded 37 ± 1% of SDZ at temperature 40 °C and pH 5 within 3 h in vitro. Strain EcN-IL with 500 mg/kg of SDZ was employed as a food supplement to feed chicken broilers, which can reduce the residue of SDZ in broiler manure by 58 ± 2% and also reduced dysbiosis of the gut microbiota due to overuse of antibiotics. The genetically engineered EcN-IL has laid a foundation for degrading SDZ in broilers and their manure.

The guanidine thiocyanate-high EDTA method for total microbial RNA extraction from severely heavy metal-contaminated soils.

Molecular analyses relying on RNA, as a direct way to unravel active microbes and their functional genes, have received increasing attention from environmental researchers recently. However, extracting sufficient and high-quality total microbial RNA from seriously heavy metal-contaminated soils is still a challenge. In this study, the guanidine thiocyanate-high EDTA (GTHE) method was established and optimized for recovering high quantity and quality of RNA from long-term heavy metal-contaminated soils. Due to the low microbial biomass in the soils, we combined multiple strong denaturants and intense mechanical lysis to break cells for increasing RNA yields. To minimize RNAase and heavy metals interference on RNA integrity, the concentrations of guanidine thiocyanate and EDTA were increased from 0.5 to 0.625 ml g-1 soil and 10 to 100 mM, respectively. This optimized GTHE method was applied to seven severely contaminated soils, and the RNA recovery efficiencies were 2.80 ~ 59.41 µg g-1 soil. The total microbial RNA of non-Cr(VI) (NT) and Cr(VI)-treated (CT) samples was utilized for molecular analyses. The result of qRT-PCR demonstrated that the expressions of two tested genes, chrA and yieF, were respectively upregulated 4.12- and 62.43-fold after Cr(VI) treatment. The total microbial RNA extracted from NT and CT samples, respectively, reached to 26.70 µg and 30.75 µg, which were much higher than the required amount (5 µg) for metatranscriptomic library construction. Besides, ratios of mRNA read were more than 86%, which indicated the high-quality libraries constructed for metatranscriptomic analysis. In summary, the GTHE method is useful to study microbes of contaminated habitats.