Using the sulfide-oxidizing bacterium Geobacillus thermodenitrificans to restrict H2S release during chicken manure composting.

Hydrogen sulfide (H2S) is a toxic gas massively released during chicken manure composting. Diminishing its release requires efficient and low cost methods. In recent years, heterotrophic bacteria capable of rapid H2S oxidation have been discovered but their applications in environmental improvement are rarely reported. Herein, we investigated H2S oxidation activity of a heterotrophic thermophilic bacterium Geobacillus thermodenitrificans DSM465, which contains a H2S oxidation pathway composed by sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO). This strain rapidly oxidized H2S to sulfane sulfur and thiosulfate. The oxidation rate reached 5.73 µmol min-1·g-1 of cell dry weight. We used G. thermodenitrificans DSM465 to restrict H2S release during chicken manure composting. The H2S emission during composting process reduced by 27.5% and sulfate content in the final compost increased by 34.4%. In addition, this strain prolonged the high temperature phase by 7 days. Thus, using G. thermodenitrificans DSM465 to control H2S release was an efficient and economic method. This study provided a new strategy for making waste composting environmental friendly and shed light on perspective applications of heterotrophic H2S oxidation bacteria in environmental improvements.

Rhodobacteraceae methanethiol oxidases catalyze methanethiol degradation to produce sulfane sulfur other than hydrogen sulfide.

Methanethiol (MT) is a sulfur-containing compound produced during dimethylsulfoniopropionate (DMSP) degradation by marine bacteria. The C-S bond of MT can be cleaved by methanethiol oxidases (MTOs) to release a sulfur atom. However, the cleaving process remains unclear, and the species of sulfur product is uncertain. It has long been assumed that MTOs produce hydrogen sulfide (H2S) from MT. Herein, we studied the MTOs in the Rhodobacteraceae family-whose members are important DMSP degraders ubiquitous in marine environments. We identified 57 MTOs from 1,904 Rhodobacteraceae genomes. These MTOs were grouped into two major clusters. Cluster 1 members share three conserved cysteine residues, while cluster 2 members contain one conserved cysteine residue. We examined the products of three representative MTOs both in vitro and in vivo. All of them produced sulfane sulfur other than H2S from MT. Their conserved cysteines are substrate-binding sites in which the MTO-S-S-CH3 complex is formed. This finding clarified the sulfur product of MTOs and enlightened the MTO-catalyzing process. Moreover, this study connected DMSP degradation with sulfane sulfur metabolism, filling a critical gap in the DMSP degradation pathway and representing new knowledge in the marine sulfur cycle field. IMPORTANCE: This study overthrows a long-time assumption that methanethiol oxidases (MTOs) cleave the C-S bond of methanethiol to produce both H2S and H2O2-the former is a strong reductant and the latter is a strong oxidant. From a chemistry viewpoint, this reaction is difficult to happen. Investigations on three representative MTOs indicated that sulfane sulfur (S0) was the direct product, and no H2O2 was produced. Finally, the products of MTOs were corrected to be S0 and H2O. This finding connected dimethylsulfoniopropionate (DMSP) degradation with sulfane sulfur metabolism, filling a critical gap in the DMSP degradation pathway and representing new knowledge in the marine sulfur cycle field.

Investigating the debrominations of a subset of brominated flame retardants by biogenic reactive sulfur species.

Brominated flame retardants (BFRs) are persistent organic pollutants. Many bacteria are able to debrominate BFRs, but the underlying mechanism is unclear. Herein, we discovered that reactive sulfur species (RSS), which have strong reductive activity and are commonly present in bacteria, might be one of the reasons leading to such ability. Experiments performed with RSS (H2S and HSSH) and BFRs indicated that RSS can debrominate BFRs via two different mechanisms simultaneously: the substitutive debromination that generates thiol-BFRs and the reductive debromination that generates hydrogenated BFRs. Debromination reactions rapidly happened under neutral pH and ambient temperature, and the debromination degree was around 30% - 55% in one hour. Two Pseudomonas strains, Pseudomonas sp. C27 and Pseudomonas putida B6-2 both produced extracellular RSS and showed debromination activity. C27 debrominated HBCD, TBECH, and TBP by 5.4%, 17.7%, and 15.9% in two days. Whereas, B6-2 debrominated the three BFRs by 0.4%, 0.6%, and 0.3% in two days. The two bacteria produced different amounts and species of RSS, which were likely responsible for the contrasted degrees of the debromination. Our finding unveiled a novel, non-enzymatic debromination mechanism that many bacteria may possess. RSS producing bacteria have potentials to contribute to bioremediation of BFRs-polluted environments.

The Pleiotropic Regulator AdpA Regulates the Removal of Excessive Sulfane Sulfur in Streptomyces coelicolor.

Reactive sulfane sulfur (RSS), including persulfide, polysulfide, and elemental sulfur (S8), has important physiological functions, such as resisting antibiotics in Pseudomonas aeruginosa and Escherichia coli and regulating secondary metabolites production in Streptomyces spp. However, at excessive levels it is toxic. Streptomyces cells may use known enzymes to remove extra sulfane sulfur, and an unknown regulator is involved in the regulation of these enzymes. AdpA is a multi-functional transcriptional regulator universally present in Streptomyces spp. Herein, we report that AdpA was essential for Streptomyces coelicolor survival when facing external RSS stress. AdpA deletion also resulted in intracellular RSS accumulation. Thioredoxins and thioredoxin reductases were responsible for anti-RSS stress via reducing RSS to gaseous hydrogen sulfide (H2S). AdpA directly activated the expression of these enzymes at the presence of excess RSS. Since AdpA and thioredoxin systems are widely present in Streptomyces, this finding unveiled a new mechanism of anti-RSS stress by these bacteria.

CD8+ T cell exhaustion and cancer immunotherapy.

Immunotherapy plays an increasingly important role in the treatment of most malignant tumors, and CD8+ T cells are the most important antitumor effector cells in the process of immunotherapy, and their number and functional status largely determine the antitumor effect. However, under continuous antigen exposure and the stimulation of inflammatory factors, CD8+ T cells gradually show a weakened proliferation and effector function, accompanied by the expression of a variety of inhibitory receptors. This state is known as CD8+ T cell "exhaustion" and often leads to the loss of control and progression of tumors. Recent studies provided us a better understanding of the mechanisms of T cell exhaustion, this review provides an overview of the activation, exhaustion mechanisms and exhaustion characteristics of CD8+ T cells. Although immunotherapy can reverse the exhaustion of CD8+ T cells and significantly improve the antitumor effects, single immunotherapy often has limitations, and it is difficult to achieve satisfactory antitumor effects, therefore, this review also summarizes up-to-date information related to cancer immunotherapy, and these emerging insights provide promising clues to the future management of malignant tumors.

A Caveat When Using Alkyl Halides as Tagging Agents to Detect/Quantify Reactive Sulfur Species.

Using alkyl halides to tag reactive sulfur species (RSSs) (H2S, per/polysulfide, and protein-SSH) is an extensively applied approach. The underlying supposition is that, as with thiols, RSS reacts with alkyl halides via a nucleophilic substitution reaction. We found that this supposition is facing a challenge. RSS also initiates a reductive dehalogenation reaction, which generates the reduced unloaded tag and oxidized RSS. Therefore, RSS content in bio-samples might be underestimated, and its species might not be precisely determined when using alkyl halide agents for its analysis. To calculate to the extent of this underestimation, further studies are still required.

Rhodanese Rdl2 produces reactive sulfur species to protect mitochondria from reactive oxygen species.

Mitochondria damage is related to a broad spectrum of pathologies including Alzheimer's, Parkinson's disease, and carcinogenesis. Recently, it has been found that reactive sulfur species (RSS) has a close connection with mitochondrial health. However, the enzyme involving in mitochondrial RSS generation and the mechanism of how RSS affects mitochondrial health are not well understood. In this study, we discovered that rhodanese 2 (Rdl2) is the main enzyme responsible for RSS generation in S. cerevisiae mitochondria, in which no sulfide:quinone oxidoreductase (Sqr) is present. Rdl2 releases sulfane sulfur atoms (S0) from stable S0 carriers (thiosulfate and dialkyl polysulfide) to produce RSS. Rdl2 deletion leads to morphological change, dysfunction, and DNA degradation of mitochondria. Rdl2-generated RSS can protect DNA from HO• attack. The reaction rate between RSS and HO• is ∼1010 M-1s-1, two magnitudes higher than that of HO• reacting with DNA. Surprisingly, hydrogen sulfide (H2S) promotes HO• production through stimulating the Fenton reaction, leading to increased DNA damage. This study highlights the antioxidation function of RSS in vivo and sheds a light on the elusive connection between RSS biogenesis and mitochondrial health.

Saccharomyces cerevisiae Rhodanese RDL2 Uses the Arg Residue of the Active-Site Loop for Thiosulfate Decomposition.

Persulfide, polysulfide and thiosulfate are examples of sulfane sulfur containing chemicals that play multiple functions in biological systems. Rhodaneses are widely present in all three kingdoms of life, which catalyze sulfur transfer among these sulfane sulfur-containing chemicals. The mechanism of how rhodaneses function is not well understood. Saccharomyces cerevisiae rhodanese 2 (RDL2) is involved in mitochondrial biogenesis and cell cycle control. Herein, we report a 2.47 Šresolution structure of RDL2 co-crystallized with thiosulfate (PDB entry: 6K6R). The presence of an extra sulfur atom Sδ, forming a persulfide bond with the Sγ atom of Cys106, was observed. Distinct from the persulfide groups in GlpE (PDB entry:1GMX) and rhobov (PDB entry:1BOI), the persulfide group of RDL2 is located in a peanut-like pocket of the neutral electrostatic field and is far away from positively charged amino acid residues of its active-site loop, suggesting no interaction between them. This finding suggests that the positively charged amino acid residues are not involved in the stabilization of the persulfide group. Activity assays indicate that the Arg111 of the active-site loop is critical for the sulfane sulfur transfer. In vitro assays indicate that Arg propels the thiosulfate decomposition. Thus, we propose that Arg can offer a hydrogen bond-rich, acidic-like microenvironment in RDL2 in which thiosulfate decomposes to release sulfane sulfur. Thr of the active-site loop of rhodaneses has the same functions as Arg. Our proposal may explain the catalyzing mechanism of rhodaneses.

Primary Two-Layered Closure of the Common Bile Duct Reduces Postoperative Bile Leakage After Laparoscopic Common Bile Duct Exploration.

Objective: The effectiveness and feasibility of the primary closure after laparoscopic common bile duct exploration (LCBDE) have been well demonstrated, however, the incidence of postoperative bile leakage after LCBDE remains high. The current study aimed at investigating whether our new suturing method could reduce the risk of bile leakage after LCBDE. Materials and Methods: This retrospective study included 81 patients who underwent primary two-layered closure or traditional primary closure of the common bile duct (CBD) after LCBDE, and the related clinical data were compared and analyzed. Results: The primary two-layered closure group had a lower rate of bile leakage compared with the traditional primary closure group (P < .05). There were no significant differences in additional parameters, such as operative time, estimated blood loss, postoperative stay duration, time to drain removal, postoperative pancreatitis, stone recurrence, and overall morbidity. No patients developed bile duct stenosis during the follow-up period. Conclusions: Primary two-layered closure of CBD can reduce the postoperative bile leakage after LCBDE. Moreover, it is a safe and effective therapeutic option for patients with choledocholithiasis.

A Red Fluorescent Protein-Based Probe for Detection of Intracellular Reactive Sulfane Sulfur.

Reactive sulfane sulfur, including persulfide and polysulfide, is a type of regular cellular component, playing an antioxidant role. Its function may be organelle-dependent; however, the shortage of probes for detecting organellar reactive sulfane sulfur has hindered further investigation. Herein, we reported a red fluorescent protein (mCherry)-based probe for specifically detecting intracellular reactive sulfane sulfur. By mutating two amino acid residues of mCherry (A150 and S151) to cysteine residues, we constructed a mCherry mutant, which reacted with reactive sulfane sulfur to form an intramolecular -Sn- bond (n ≥ 3). The bond largely decreased the intensity of 610 nm emission (excitation at 587 nm) and slightly increased the intensity of 466 nm emission (excitation at 406 nm). The 466/610 nm emission ratio was used to indicate the relative abundance of reactive sulfane sulfur. We then expressed this mutant in the cytoplasm and mitochondria of Saccharomyces cerevisiae. The 466/610 nm emission ratio revealed that mitochondria had a higher level of reactive sulfane sulfur than cytoplasm. Thus, the mCherry mutant can be used as a specific probe for detecting reactive sulfane sulfur in vivo.

Construction of Escherichia coli Whole-Cell Biosensors for Statin Efficacy and Production Test.

Statins are widely used cholesterol-lowering drugs. Their potential application in anti-cancer treatment is also under investigation. The individual variance in statin response has been observed, which may be caused by the variation in human HMG-CoA reductase (hHMGR)-the inhibition target of statin drugs. Herein, we reported the design and construction of two Escherichia coli whole-cell biosensors. The first one is statin-efficacy testing sensor, which is composed of two separate modules: a hybrid mevalonate (MVA) pathway and a HMG-CoA sensing system. A truncated hHMGR was used as the key enzyme of the MVA pathway and a promiscuous transcription factor (TF) BsFapR was used as the HMG-CoA sensor. When hHMGR was inhibited by statins, HMG-CoA accumulated intracellularly and was sensed by BsFapR, which subsequently turned on its cognate promoter. This biosensor has the potential to be used as a "precision medicine" tool-selecting potent statin drugs for individual patients. The second one is a statin-production testing sensor, which is based on another promiscuous TF AraCM that can sense statins. This biosensor can be used in optimization of statin-producing strains. The prototypes of these two biosensors were successfully constructed and their further optimization is highly expected.

Synthetic Gene Circuits Enable Escherichia coli To Use Endogenous H2S as a Signaling Molecule for Quorum Sensing.

Microorganisms often use specific autoinducers other than common metabolites for quorum sensing (QS). Herein, we demonstrated that Escherichia coli produced sulfide (H2S, HS-, and S2-) with the concentrations proportionally correlated to its cell density. We then designed synthetic gene circuits that used H2S as an autoinducer for quorum sensing. A sulfide/quinone oxidoreductase converted diffusible H2S to indiffusible hydrogen polysulfide (HSnH, n ≥ 2), and a gene regulator CstR sensed the latter to turn on the gene expression. We constructed three element libraries, with which 24 different circuits could be assembled for adjustable sensitivity to cell density. The H2S-mediated gene circuits endowed E. coli cells within the same batch or microcolony with highly synchronous behaviors. Using them we successfully constructed cell factories capable of an autonomous switch from growth phase to production phase. Thus, these circuits provide a new tool-kit for metabolic engineering and synthetic biology.

Using resonance synchronous spectroscopy to characterize the reactivity and electrophilicity of biologically relevant sulfane sulfur.

Sulfane sulfur is common inside cells, playing both regulatory and antioxidant roles. However, there are unresolved issues about its chemistry and biochemistry. We report the discovery that reactive sulfane sulfur such as polysulfides and persulfides could be detected by using resonance synchronous spectroscopy (RS2). With RS2, we showed that inorganic polysulfides at low concentrations were unstable with a half-life about 1 min under physiological conditions due to reacting with glutathione. The protonated form of glutathione persulfide (GSSH) was electrophilic and had RS2 signal. GSS- was nucleophilic, prone to oxidation, but had no RS2 signal. Using this phenomenon, pKa of GSSH was determined as 6.9. GSSH/GSS- was 50-fold more reactive than H2S/HS- towards H2O2 at pH 7.4, supporting reactive sulfane sulfur species like GSSH/GSS- may act as antioxidants inside cells. Further, protein persulfides were shown to be in two forms: at pH 7.4 the deprotonated form (R-SS-) without RS2 signal was not reactive toward sulfite, and the protonated form (R-SSH) in the active site of a rhodanese had RS2 signal and readily reacted with sulfite to produce thiosulfate. These data suggest that RS2 of sulfane sulfur is likely associated with its electrophilicity. Sulfane sulfur showed species-specific RS2 spectra and intensities at physiological pH, which may reveal the relative abundance of a reactive sulfane sulfur species inside cells.

Effect of GnT-V knockdown on the proliferation, migration and invasion of the SMMC7721/R human hepatocellular carcinoma drug-resistant cell line.

Hepatocellular carcinoma (HCC) is a commonly occurring malignant tumor, with a high incidence rate. The present study aimed to investigate the effect of knocking down the N­glycosyltransferase­V (GnT­V) protein on the proliferation, migration and invasion of the human HCC drug­resistant cell line, SMMC7721/R. SMMC7721/R cells with GnT­V­knockdown (SMMC­7721/R­GnT­V) were constructed using the method of lentiviral transfection. The expression of GnT­V was assessed using reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting. Cell proliferation was determined using an MTT assay, and the extent of cellular apoptosis was assessed using flow cytometric analysis. Additionally, the metastatic ability of the cells in vitro was analyzed using cell adhesion and invasion assays. Western blotting was used to investigate the protein expression levels of caspase­3, caspase­9, Bcl­2, Bax, matrix metalloproteinase (MMP)­2 and MMP­9, and RT­qPCR was used to determine the mRNA expression levels of the genes for the breast cancer resistance protein and P­glycoprotein in the SMMC­7721/R cells. Taken together, the results of the present study revealed that the knockdown of GnT­V significantly suppressed the proliferation, migration and invasion (P<0.05) of the SMMC­7721/R cells. Furthermore, the possible mechanism underlying these phenomena may be associated with the induction of mitochondria­mediated apoptosis, inhibition of the degradation of the extracellular matrix and an enhancement of the drug-sensitivity. GnT­V­knockdown may therefore be used to treat drug­resistant HCC in the future.

Interaction of Apolipoprotein Model Peptides with Liposomes: A High Performance Liquid Chromatography Study.

The apparent partition constants of two amphiphilic peptides, Amp1 and Amp2, for partitioning into phosphatidylglycerol/phosphatidylcholine bilayer were measured using size-exclusion high performance liquid chromatograph. The exposed amino groups of vesicle-bound peptides were studied by TNBS assay. It was proposed that their N-terminals were exposed to the aqueous phase, and that the main explanation for the stronger interaction of Amp1 with lipid bilayer compared with Amp2 was its stronger lipid binding ability, though Amp1 was also buried deeper in the lipid membrane. It was also found that the two peptides were polymerized in buffer, with their amino groups almost totally buried within the polymers.

Interaction of the Apolipoprotein A-I Model Peptides with Liposomes.

Two amphiphilic peptides, Amp1 and Amp2, were synthesized according to the sequence of the lipid-binding domain in apolipoprotein. Amp2 has a Val residue substituted for the Lys at the 4th position of Amp1. Intrinsic fluorescence spectra, peptide-induced leakage of calcein-laoded liposomes, quenching of tryptophan fluorescene by iodide and acrylamide, circular dichroism spectra, and measurement of the membrane penetration depth of tryptophan residue with spin-labeled phospholipids indicate unexceptionally that Amp1 interacted more strongly with the lipid bilayer than Amp2. It is proposed that class A amphiphilic alpha-helix is buried in the membrane in such a way that its long axis is oriented parallel to the membrane plane, and the electrostatic interaction between the positively charged residues located at the polar-nonpolar interface of the amphiphilic helix with the negatively charged head groups of phospholipids is important to the lipid affinity of the amphiphilic peptide.

Influence of Lipid Composition on the Interaction of Apolipoprotein Model Peptides with Liposomes.

Two amphiphilic peptides, Ampl and Amp2, were synthesized according to the sequence of the lipid-binding domain in apoliporotein. Amp2 has a Val residue substituted for the Lys at the 4th position of Ampl. Interaction between Ampl / Amp2 and liposomes with different lipid compositions were compared by studying the blue shifts of the intrinsic fluorescence emission maxima, the peptide-induced lipsome leakage and the quenching of tryptophan fluorescence by acrylamide. The influence of temperature on interactions was studied as well. Ampo1 interacted stronger with acidic lipids while Amp2 interacted stronger with zwitterionic lipids. The interaction was reinforced with the increasing extent of lipid unsaturation as well as with the increase of temperature. The lipid unsaturation had amore prominent effect.