Exploiting tertiary lymphoid structures gene signature to evaluate tumor microenvironment infiltration and immunotherapy response in colorectal cancer.

Background: Tertiary lymphoid structures (TLS) is a particular component of tumor microenvironment (TME). However, its biological mechanisms in colorectal cancer (CRC) have not yet been understood. We desired to reveal the TLS gene signature in CRC and evaluate its role in prognosis and immunotherapy response. Methods: The data was sourced from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Based on TLS-related genes (TRGs), the TLS related subclusters were identified through unsupervised clustering. The TME between subclusters were evaluated by CIBERSORT and xCell. Subsequently, developing a risk model and conducting external validation. Integrating risk score and clinical characteristics to create a comprehensive nomogram. Further analyses were conducted to screen TLS-related hub genes and explore the relationship between hub genes, TME, and biological processes, using random forest analysis, enrichment and variation analysis, and competing endogenous RNA (ceRNA) network analysis. Multiple immunofluorescence (mIF) and immunohistochemistry (IHC) were employed to characterize the existence of TLS and the expression of hub gene. Results: Two subclusters that enriched or depleted in TLS were identified. The two subclusters had distinct prognoses, clinical characteristics, and tumor immune infiltration. We established a TLS-related prognostic risk model including 14 genes and validated its predictive power in two external datasets. The model's AUC values for 1-, 3-, and 5-year overall survival (OS) were 0.704, 0.737, and 0.746. The low-risk group had a superior survival rate, more abundant infiltration of immune cells, lower tumor immune dysfunction and exclusion (TIDE) score, and exhibited better immunotherapy efficacy. In addition, we selected the top important features within the model: VSIG4, SELL and PRRX1. Enrichment analysis showed that the hub genes significantly affected signaling pathways related to TLS and tumor progression. The ceRNA network: PRRX1-miRNA (hsa-miR-20a-5p, hsa-miR-485-5p) -lncRNA has been discovered. Finally, IHC and mIF results confirmed that the expression level of PRRX1 was markedly elevated in the TLS- CRC group. Conclusion: We conducted a study to thoroughly describe TLS gene signature in CRC. The TLS-related risk model was applicable for prognostic prediction and assessment of immunotherapy efficacy. The TLS-hub gene PRRX1, which had the potential to function as an immunomodulatory factor of TLS, could be a therapeutic target for CRC.

Comparative Metagenomic and Metatranscriptomic Analyses Reveal the Response of Black Soldier Fly (Hermetia illucens) Larvae Intestinal Microbes and Reduction Mechanisms to High Concentrations of Tetracycline.

Black soldier fly (Hermetia illucens L) larvae (BSFL) possess remarkable antibiotic degradation abilities due to their robust intestinal microbiota. However, the response mechanism of BSFL intestinal microbes to the high concentration of antibiotic stress remains unclear. In this study, we investigated the shift in BSFL gut microbiome and the functional genes that respond to 1250 mg/kg of tetracycline via metagenomic and metatranscriptomic analysis, respectively. The bio-physiological phenotypes showed that the survival rate of BSFL was not affected by tetracycline, while the biomass and substrate consumption of BSFL was slightly reduced. Natural BSFL achieved a 20% higher tetracycline degradation rate than the germ-free BSFL after 8 days of rearing. Metagenomic and metatranscriptomic sequencing results revealed the differences between the entire and active microbiome. Metatranscriptomic analysis indicated that Enterococcus, Vagococcus, Providencia, and Paenalcaligenes were the active genera that responded to tetracycline. Furthermore, based on the active functional genes that responded to tetracycline pressure, the response mechanisms of BSFL intestinal microbes were speculated as follows: the Tet family that mediates the expression of efflux pumps expel tetracycline out of the microbes, while tetM and tetW release it from the ribosome. Eventually, tetracycline was degraded by deacetylases and novel enzymes. Overall, this study provides novel insights about the active intestinal microbes and their functional genes in insects responding to the high concentration of antibiotics.

Biochar Can Improve Absorption of Nitrogen in Chicken Manure by Black Soldier Fly.

(1) Background: There is growing interest in using insects to treat nutrient-rich organic wastes, such as the black soldier fly (BSF), one of the most efficient organic waste recyclers for upcycling nutrients into the food system. Although biochar (BC) was shown to enhance nutrient retention and the final product quality during the composting of livestock and poultry manure in many previous studies, little information is available on the effect of BC on livestock manure bioconversion by black soldier fly larvae (BSFL). (2) Methods: This study investigated the effect of adding a small amount of BC to chicken manure (CM) on the bioconversion system of the black soldier fly (including N2O and NH3 emissions and the final distribution of nitrogen during the treatment process). (3) Results: The lowest N2O and NH3 emission and highest residual nitrogen in the substrate were observed in the 15% BC treatment. The highest bioconversion rate of CM (8.31%) and the peak of larval biomass was obtained in the 5% BC treatment. (4) Conclusions: The results demonstrate the feasibility of adding 5% BC to reduce pollution and achieve a satisfactory BSFL-based CM bioconversion efficiency.

Bacillus velezensis EEAM 10B Strengthens Nutrient Metabolic Process in Black Soldier Fly Larvae (Hermetia illucens) via Changing Gut Microbiome and Metabolic Pathways.

Insects are a potential alternative protein source to solve the food shortage crisis. Previous studies have illustrated that probiotics can improve the substrate conversion efficiency of insects and increase insect protein content. However, the effects of probiotics on insect physiology and nutrient metabolism are still not well understood. Here, the black soldier fly larvae (BSFL), Hermetia illucens (Diptera: Stratiomyidae), was used as a study subject to deeply investigate the specific interaction among a novel probiotic, Bacillus velezensis EEAM 10B (10B), intestinal microbiota, and the host. In this study, the effects of 10B on the survival and physiology of BSFL were first analyzed. It shows that 10B significantly elevated the substrate conversion rate, average dry weight, and protein content of BSFL by 5%, 0.13 g/pc, and 8%, respectively. Then, we assessed the effect of 10B on the microbial community composition in the gut and frass of BSFL using Illumina Miseq sequencing. It shows that 10B significantly altered the microbial composition of the gut, but not that of the frass. Pearson's correlation analysis further showed that the Bacillus, unclassified_of_Caloramatoraceae, and Gracilibacillus were positively correlated with the survival rate, crude protein content, and substrate conversion rate of BSFL. To further investigate the effect of 10B on host metabolism, metabolic analyses on germ-free BSFL, monobacterial intestinal BSFL, and natural BSFL were also performed. The results proved that 10B (i) played a vital role in the survival of BSFL; and (ii) regulated the amino acid synthetic and metabolic process of BSFL, thus leading to the rise of the protein content of BSFL. In addition, vitamin backfill assays verified that the BSFL survival rate was significantly improved by supplying the germ-free BSFL with riboflavin, which further suggests that 10B determines the survival of BSFL via delivering riboflavin. Overall, this study provides a reference for understanding the comprehensive contribution of a specific probiotic to its host.

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.

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.

Exploring novel Cr(VI) remediation genes for Cr(VI)-contaminated industrial wastewater treatment by comparative metatranscriptomics and metagenomics.

Microbial remediation is a promising method to treat Cr(VI) in industrial wastewater. The remediation efficiency and stress-resistance ability of Cr(VI) remediation genes in microbes are the limiting factors for their application in industrial wastewater treatment. To screen novel highly efficient Cr(VI) remediation genes, comparative metatranscriptomic and metagenomic analyses were performed on long-term Cr(VI)-contaminated riparian soil with/without additional Cr(VI) treatment. The most suitable Cr(VI) treatment time was determined to be 30 min according to the high quality RNA yield and fold changes in gene expression. Six novel genes, which had complete open reading frames (ORFs) in metagenomic libraries, were identified from unculturable microbes. In the phenotypic functional assay, all novel genes enhanced the Cr(VI) resistance/reduction ability of E. coli. In the industrial wastewater treatment, E-mcr and E-gsr presented at least 50% Cr(VI) removal efficiencies in the presence of 200-600 µM of Cr(VI), without a decrease in efficiency over 17 days. The stress resistance assay showed that gsr increased the growth rate of E. coli by at least 30% under different extreme conditions, and thus, gsr was identified as a general stress-response gene. In the Cr valence distribution assay, E-mcr presented ~40 µM higher extracellular Cr (III) compared to E-yieF. Additionally, transmission electron microscopy (TEM) of E-mcr showed bulk black agglomerates on the cell surface. Thus, mcr was identified as a membrane chromate reductase gene. This research provides a new idea for studying novel highly efficient contaminant remediation genes from unculturable microbes.

Immobilized-microbial bioaugmentation protects aerobic denitrification from heavy metal shock in an activated-sludge reactor.

The inhibition of denitrification by heavy metals is a problem in nitrogen wastewater treatment, but the solutions are rarely studied. In this study, Pseudomonas brassicacearum LZ-4, immobilized in sodium alginate-kaolin, was applied in an activated-sludge reactor to protect denitrifiers from hexavalent chromium (Cr(VI)). Q-PCR result showed that the strain LZ-4 was incorporated into activated sludge under the help of immobilization. In the non-bioaugmentation system, the removal efficiency of nitrate was decreased by 86.07% by 30 mg/L Cr(VI). Whereas, denitrification was protected and 95% of nitrate was removed continuously in immobilized-cell bioaugmentation system. Miseq sequencing data showed that bioaugmentation decreased the impact of Cr(VI) on microbial communities and increased the abundance of denitrifiers. Based on the results of biomass and extracellular polymers, activated sludge was protected from Cr(VI) toxicity. This discovery will provide a feasible technique for nitrogen wastewater treatment in the presence of distressing heavy metals.

Microbial communities respond to microenvironments in lungs of mice under simulated exposure to cadmium aerosols.

Inhalable pollutants are inducing factors of lung diseases and have been widely studied. Previous studies described imbalances in pulmonary microbial communities and representatively predominant microorganisms in clinical specimens of individuals with lung diseases. However, the direct effect of inhalable pollutants on pulmonary microorganisms has not been determined to date. Cadmium is a common inhalable pollutant from manufacturing activities, and its effect on pulmonary microorganisms was investigated in this study. Such techniques as optical respiratory plethysmography, high-throughput pulmonary histological assessment and differential centrifugation were used to characterize pulmonary microenvironments, and high-throughput sequencing was used to analyze pulmonary microbial diversity. We found variations in pulmonary microenvironmental factors, such as air supply level, nutrition and inflammatory stress. Under inhalable cadmium exposure at different doses, pulmonary microorganisms were differentially subjected and sensitive to various microenvironmental stresses (e.g., inflammation, pH, ventilation, nutrition and related changes of lung tissue structure) and might participate in microenvironmental remodeling, such as pneumonia and pulmonary fibrosis. Inflammatory stress and Lactobacillus were the main microenvironmental factor and susceptible microorganism, respectively. The various pulmonary microenvironments influenced the metabolisms of pulmonary microbial communities, presenting differences in microbial collinearities, gene function levels and metabolic pathway levels among groups.

A novel biosensor for zinc detection based on microbial fuel cell system.

Microbial fuel cell (MFC) biosensors are self-sustainable device for monitoring of various substrates; however, for heavy metals detection are still scarce. In this study, E. coli BL21 was engineered to express the zntR, ribB, and oprF genes with PzntA promoter, which could sense zinc (Zn2+) for riboflavin and porin production. The engineered strain produced high levels of riboflavin (2.4-3.6 µM) and improved cell membrane permeability, with a positive correlation of Zn2+ (0-400 µM). The strain was then employed in MFC biosensor under the following operational parameters: external resistance 1000 Ω, pH 9, and temperature 37 °C for Zn2+ sensing. The maximum voltages (160, 183, 260, 292, and 342 mV) of the constructed MFC biosensor have a linear relationship with Zn2+ concentrations (0, 100, 200, 300, and 400 µM, respectively) (R2 = 0.9777). An Android App was developed for the biosensor system that could sense Zn2+ in real-time and in situ. The biosensor was applied to wastewater with different Zn2+ concentrations and the results showed that the detection range for Zn2+ was 20-100 µM, which covers common Zn2+ safety standards. The results obtained with developed MFC biosensor were comparable to conventional methods such as colorimetric, flame atomic absorption spectroscopy (FAAS), and inductively coupled plasma optical emission spectroscopy (ICP-OES). In summary, MFC biosensor with biosynthetic strain is an efficient and affordable system for real-time monitoring and sensing of heavy metals.

Micro-aeration in anode chamber promotes p-nitrophenol degradation and electricity generation in microbial fuel cell.

Biodegradation of recalcitrant organic compounds in microbial fuel cell (MFC) is limited, due to its strong electron affinity and persisted in anaerobic condition. In this study, Pseudomonas monteilii LZU-3 degraded p-nitrophenol (PNP) and generated current at 100 mg L-1 of PNP in anode MFC with the addition of oxygen. The highest PNP degradation was 4, 37.75, and 99.89% in anaerobic, aerobic, and aerated anode of MFC respectively, at 7 h. The maximum voltage generation in aerated anode was 183 mV, which was comparatively higher than aerobic (150 mV) and anaerobic (68 mV). The qRT-PCR results confirmed that the oxygenase genes in strain LZU-3 were up-regulated from 17.51 to 39.39-fold at 1.6-4.5 mg L-1 of oxygen concentrations resulted in PNP degradation in anode MFC. This study demonstrated that supplementation of oxygen into the anode MFC might be a potential approach for biodegradation of recalcitrant compounds and electricity generation.

Microbial Community Structure and Function Indicate the Severity of Chromium Contamination of the Yellow River.

The Yellow River is the most important water resource in northern China. In the recent past, heavy metal contamination has become severe due to industrial processes and other anthropogenic activities. In this study, riparian soil samples with varying levels of chromium (Cr) pollution severity were collected along the Gansu industrial reach of the Yellow River, including samples from uncontaminated sites (XC, XGU), slightly contaminated sites (LJX, XGD), and heavily contaminated sites (CG, XG). The Cr concentrations of these samples varied from 83.83 mg⋅kg-1 (XGU) to 506.58 mg⋅kg-1 (XG). The chromate [Cr (VI)] reducing ability in the soils collected in this study followed the sequence of the heavily contaminated > slightly contaminated > the un-contaminated. Common Cr remediation genes chrA and yieF were detected in the XG and CG samples. qRT-PCR results showed that the expression of chrA was up-regulated four and threefold in XG and CG samples, respectively, whereas the expression of yieF was up-regulated 66- and 7-fold in the same samples after 30 min treatment with Cr (VI). The copy numbers of chrA and yieF didn't change after 35 days incubation with Cr (VI). The microbial communities in the Cr contaminated sampling sites were different from those in the uncontaminated samples. Especially, the relative abundances of Firmicutes and Bacteroidetes were higher while Actinobacteria was lower in the contaminated group than uncontaminated group. Further, potential indicator species, related to Cr such as Cr-remediation genera (Geobacter, PSB-M-3, Flavobacterium, and Methanosarcina); the Cr-sensitive genera (Skermanella, Iamia, Arthrobacter, and Candidatus Nitrososphaera) were also identified. These data revealed that Cr shifted microbial composition and function. Further, Cr (VI) reducing ability could be related with the expression of Cr remediation genes.