Disruption of Plin5 degradation by CMA causes lipid homeostasis imbalance in NAFLD.

BACKGROUND & AIMS: The pathological hallmark of nonalcoholic fatty liver disease (NAFLD) is an imbalance in hepatic lipid homeostasis, in which lipophagy has been found to play a vital role. However, the underlying molecular mechanisms remain unclear. We investigated the role of chaperone-mediated autophagy (CMA) in the pathogenesis of NAFLD. METHODS: CMA activity was evaluated in liver tissues from NAFLD patients and high-fat diet (HFD)-fed mice. Liver-specific LAMP2A-knockout mice and HepG2 cells lacking LAMP2A [L2A(-) cells] were used to investigate the influence of CMA on lipolysis in hepatocytes. The expression of Plin5, a lipid droplet (LD)-related protein, was also evaluated in human and mouse liver tissues and in [L2A(-)] cells. RESULTS: Here, we found disrupted CMA function in the livers of NAFLD patients and animal models, displaying obvious reduction of LAMP2A and concurrent with decreased levels of CMA-positive regulators. More LDs and higher serum triglycerides accumulated in liver-specific LAMP2A-knockout mice and L2A(-) cells under high-fat challenge. Meanwhile, deleting LAMP2A hindered LD breakdown but not increased LD formation. In addition, the LD-associated protein Plin5 is a CMA substrate, and its degradation through CMA is required for LD breakdown. CONCLUSIONS: We propose that the disruption of CMA-induced Plin5 degradation obstacles LD breakdown, explaining the lipid homeostasis imbalance in NAFLD.

Birth preparation acupuncture for normalising birth: An analysis of NHS service routine data and proof of concept.

A number of studies show that acupuncture may help with labour and delivery. An NHS maternity acupuncture service providing birth preparation acupuncture has assessed its routine hospital maternity annual data from 2014 to 2016 to see what effect it had on labour and delivery outcomes. The data from this service was analysed and women who had birth preparation acupuncture were compared with those who did not receive it. Maternal age, parity and socio-economic status were considered confounders and were adjusted for in the analysis. Women who received acupuncture had more normal births (less surgical births) [OR 0.76 (0.64, 0.91)], required less intrapartum analgesia [OR 0.74 (0.63, 0.86)], fewer components of an induction of labour [OR 0.74 (0.61, 0.91)] and a reduced length of a hospital stay [OR 0.91 (0.87, 0.95)]. The patients highly valued the availability of acupuncture within the maternity service as it enhanced their patient journey.Impact statementWhat is already known on this subject? Numerous studies provide evidence for the effects of acupuncture in normalising pregnancy and birth. These effects include musculoskeletal preparation of the pelvis, cervical ripening, enhancing endogenous oxytocin release, and analgesic properties.What do the results of this study add? Our analysis shows that women who received birth preparation acupuncture had fewer surgical births, required less intrapartum analgesia, less components of induction of labour and had a reduced length of hospital stay, supporting the use of maternity acupuncture in normalising birth outcomes.What are the implications of these findings for clinical practice and/or further research? The findings show that acupuncture, by potentially normalising birth, may lead to reductions in costs of service. Further, additional research is required to see whether acupuncture is cost effective and could have an adjunctive role as a complementary therapy for improving birth outcomes and a woman's experience of childbirth.

SLC27A2 regulates miR-411 to affect chemo-resistance in ovarian cancer.

Although platinum-based chemotherapies have long been used as standard treatment in ovarian cancer, cisplatin resistance is a major problem that restricts its use. Herein, we investigate the biological function of SLC27A2 and its underlying mechanisms in regulating chemo-resistance in ovarian cancer. The findings show that SLC27A2 down-regulation in primary ovarian cancer tissues correlates with chemo-resistance and poor patient survival in our patient cohort. Significantly, we demonstrate that up-regulation of SLC27A2 by lentivirus-mediated p-SLC27A2 sensitizes ovarian cancer cells to cisplatin in vitro and in vivo via apoptosis. Mechanistic investigation reveals that miR-411 is the most strikingly over-expressed gene in response to ectopic expression of SLC27A2, but under-expressed in recurrent ovarian cancer tissues. Lower miR-411 expression contributes to ovarian cancer chemo-resistance in vitro and in vivo. Furthermore, SLC27A2 directly binds specific sites in the miR-411 promoter region and promoter activity decreases after mutation of putative SLC27A2-binding sites. This indicates that SLC27A2 is required for the transcriptional induction of miR-411. The luciferase assays also confirm that miR-411 directly targets ABCG2 in ovarian cancer, and overall findings establish the SLC27A2-miR-411-ABCG2 pathway in the regulation of ovarian cancer chemo-resistance with potential therapeutic applications.

Genome-wide association studies identify susceptibility loci affecting respiratory disease in Chinese Erhualian pigs under natural conditions.

Prevalence of swine respiratory disease causes poor growth performance in and serious economic losses to the swine industry. In this study, a categorical trait of enzootic pneumonia-like (EPL) score representing the infection gradient of a respiratory disease, more likely enzootic pneumonia, was recorded in a herd of 332 Chinese Erhualian pigs. According to their EPL scores and the disease effect on weight gains, these pigs were grouped into controls (EPL score ≤ 1) and cases (EPL score > 1). The weight gain of the case group reduced significantly at days 180, 210, 240 and 300 as compared to the control group. The heritability of EPL score was estimated to be 0.24 based on the pedigree information using a linear mixed model. All 332 Erhualian pigs and their nine sire parents were genotyped with Illumina Porcine 60K SNP chips. Two genome-wide association studies were performed under a generalized linear mixed model and a case-control model respectively. In total, five loci surpassed the suggestive significance level (P = 2.98 × 10-5 ) on chromosomes 2, 8, 12 and 14. CXCL6, CXCL8, KIT and CTBP2 were highlighted as candidate genes that might play important roles in determining resistance/susceptibility to swine EP-like respiratory disease. The findings advance understanding of the genetic basis of resistance/susceptibility to respiratory disease in pigs.

Microarray gene expression profiling for identifying different responses to radiotherapy and chemoradiotherapy in patients with cervical cancer.

PURPOSE: Cervical cancer, which is treated by radiotherapy (RT) and chemoradiotherapy (CRT), has high morbidity and mortality in women. This study aimed to identify differences in gene response to CRT and RT. MATERIALS AND METHODS: GSE3578 was downloaded from Gene Expression Omnibus including specimens from 20 RT-treated patients and 19 CRT-treated patients. Differentially expressed genes (DEGs) were identified using siggenes package in R. Protein-protein interaction (PPI) network was visualized by cytoscape. MCODE and cytoscape was used separately to mine and construct modules in the PPI network. Transcription factor (TF)-DEG and miRNA-DEG pairs were predicted and then visualized by cytoscape. RESULTS: Total 22 upregulated and 181 downregulated genes were identified in CRT samples. Several functions were enriched for these DEGs. A module involving ZNF449 and ZNF673 was mined from the PPI network of downregulated genes. In the TF-DEG regulatory networks, downregulated GATA3 (which was modulated by SPI) was also a TF, as well as upregulated CDK6 was regulated by several TFs (e.g. GATA3). Hsa-miR-17, hsa-miR-34a, hsa-miR-124, hsa-miR-I185-2-3p, hsa- 1185-1-3p, and hsa-let-7f-2-3p were identified as key miRNAs in the miRNA-DEG regulatory network. Conclu- sion: CRT might cure cervical cancer by acting on those molecules that were more sensitive to CRT than CT.

Secondary growth mechanism of SiGe islands deposited on a mixed-phase microcrystalline Si by ion beam co-sputtering.

We discuss the SiGe island co-sputtering deposition on a microcrystalline silicon (µc-Si) buffer layer and the secondary island growth based on this pre-SiGe island layer. The growth phenomenon of SiGe islands on crystalline silicon (c-Si) is also investigated for comparison. The pre-SiGe layer grown on µc-Si exhibits a mixed-phase structure, including SiGe islands and amorphous SiGe (a-SiGe) alloy, while the layer deposited on c-Si shows a single-phase island structure. The preferential growth and Ostwald ripening growth are shown to be the secondary growth mechanism of SiGe islands on µc-Si and c-Si, respectively. This difference may result from the effect of amorphous phase Si (AP-Si) in µc-Si on the island growth. In addition, the Si-Ge intermixing behavior of the secondary-grown islands on µc-Si is interpreted by constructing the model of lateral atomic migration, while this behavior on c-Si is ascribed to traditional uphill atomic diffusion. It is found that the aspect ratios of the preferential-grown super islands are higher than those of the Ostwald-ripening ones. The lower lateral growth rate of super islands due to the lower surface energy of AP-Si on the µc-Si buffer layer for the non-wetting of Ge at 700 °C and the stronger Si-Ge intermixing effect at 730 °C may be responsible for this aspect ratio difference.

Electronic properties of single Ge/Si quantum dot grown by ion beam sputtering deposition.

The dependence of the electronic properties of a single Ge/Si quantum dot (QD) grown by the ion-beam sputtering deposition technique on growth temperature and QD diameter is investigated by conductive atomic force microscopy (CAFM). The Si-Ge intermixing effect is demonstrated to be important for the current distribution of single QDs. The current staircase induced by the Coulomb blockade effect is observed at higher growth temperatures (>700 °C) due to the formation of an additional barrier between dislocated QDs and Si substrate for the resonant tunneling of holes. According to the proposed single-hole-tunneling model, the fact that the intermixing effect is observed to increase as the incoherent QD size decreases may explain the increase in the starting voltage of the current staircase and the decrease in the current step width.

Evaluation of the genetic diversity of Bupleurum using amplified fragment length polymorphism analysis.

Radix bupleuri (Chaihu), the dried root of the Bupleurum plant, is an important component of traditional Chinese medicine. In this study, we examined the genetic diversity of 11 Bupleurum strains, originating from 7 provinces in China, using amplified fragment length polymorphism analysis. A total of 274 polymorphic bands were obtained using 6 primer combinations, indicating a high level of polymorphism across all strains. An estimation of the relative relationships among strains revealed genetic distances ranging from 0.2183 to 0.7372, with an average of 0.4161. The 2 most closely related varieties were Bupleurum chinense DC. strains collected from Lushi, Henan, and Zhangjiakou, Hebei, with a genetic nearness of 0.2183. Hierarchical clustering divided the strains into 3 main groups, with B. falcatum L. from Hebei and Liaoning Provinces forming a cluster that diverged from that of B. smithii Wolff. and B. chinense DC. B. falcatum L. (Sandao chaihu), collected from Heze, Shandong, clustered independently of the other strains, suggesting that this strain may have been introduced from a different location or that it arose as a result of intraspecific variation. B. smithii Wolff. (Hei chaihu) was closely associated with B. scorzonerifolium Willd. (Nan chaihu) and B. chinense DC. (Bei chaihu), suggesting a common genetic origin.

Differential regulation of interleukin-12 (IL-12)/IL-23 by Tim-3 drives T(H)17 cell development during hepatitis C virus infection.

Cytokine production by innate immunity is critical for shaping the adaptive immunity through regulation of T cell differentiation. In this report, we studied T cell immunoglobulin mucin domain protein 3 (Tim-3) expression on monocytes and its regulatory effect on interleukin-12 (IL-12)/IL-23 production by CD14(+) monocytes, as well as IL-17 production by CD4(+) T cells in individuals with chronic hepatitis C virus (HCV) infection. We found that Tim-3 and IL-23p19 are highly expressed and that IL-12p35 is inhibited in human CD14(+) monocytes, while IL-17 expression is upregulated in CD4(+) T cells, in chronically HCV-infected individuals compared to healthy subjects. Interestingly, Tim-3 expression is closely associated with the differential regulation of IL-12/IL-23 expression in CD14(+) monocytes and correlated to IL-17 production by CD4(+) T cells. These Tim-3-associated IL-12/IL-23/IL-17 dysregulations in HCV-infected individuals are also recapitulated in vitro by incubating healthy monocytes or peripheral blood mononuclear cells with Huh-7 hepatoma cells transfected with HCV RNA. Importantly, blocking Tim-3 signaling on monocytes restores the balance of IL-12/IL-23 through the intracellular STAT3 signaling, which in turn reverses the upregulated IL-17 expression both ex vivo and in vitro. Our findings suggest that Tim-3-mediated differential regulation of IL-12/IL-23 drives T(H)17 cell development, a milieu favoring viral persistence and autoimmune phenomenon during HCV infection.

Tim-3 pathway controls regulatory and effector T cell balance during hepatitis C virus infection.

Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. Upregulation of inhibitory signaling pathways (such as T cell Ig and mucin domain protein-3 [Tim-3]) and accumulation of regulatory T cells (Tregs) play pivotal roles in suppressing antiviral effector T cell (Teff) responses that are essential for viral clearance. Although the Tim-3 pathway has been shown to negatively regulate Teffs, its role in regulating Foxp3(+) Tregs is poorly explored. In this study, we investigated whether and how the Tim-3 pathway alters Foxp3(+) Treg development and function in patients with chronic HCV infection. We found that Tim-3 was upregulated, not only on IL-2-producing CD4(+)CD25(+)Foxp3(-) Teffs, but also on CD4(+)CD25(+)Foxp3(+) Tregs, which accumulate in the peripheral blood of chronically HCV-infected individuals when compared with healthy subjects. Tim-3 expression on Foxp3(+) Tregs positively correlated with expression of the proliferation marker Ki67 on Tregs, but it was inversely associated with proliferation of IL-2-producing Teffs. Moreover, Foxp3(+) Tregs were found to be more resistant to, and Foxp3(-) Teffs more sensitive to, TCR activation-induced cell apoptosis, which was reversible by blocking Tim-3 signaling. Consistent with its role in T cell proliferation and apoptosis, blockade of Tim-3 on CD4(+)CD25(+) T cells promoted expansion of Teffs more substantially than Tregs through improving STAT-5 signaling, thus correcting the imbalance of Foxp3(+) Tregs/Foxp3(-) Teffs that was induced by HCV infection. Taken together, the Tim-3 pathway appears to control Treg and Teff balance through altering cell proliferation and apoptosis during HCV infection.

Heme oxygenase-1: a molecular brake on hepatocellular carcinoma cell migration.

Hepatocellular carcinoma (HCC) is a fatal disease with great public health impact worldwide. Heme oxygenase (HO)-1 has recently been reported as an important player in tumor angiogenesis and metastasis. However, the role of HO-1 in liver cancer metastasis is unclear. In this study, we explored genetic differences and downstream signal transduction pathways of HO-1 in liver cancer cell lines. HO-1 wild-type and mutant cell lines were generated from human liver cancer cell line HepG2. The overexpression of wild-type HO-1 decreased the migration of HepG2 cells. In contrast, the overexpression of mutant HO-1G143H increased the migration of the cancer cells. Interleukin (IL)-6 is one of the major downstream molecules that mediated this process because IL-6 expression and migration are suppressed by HO-1 and increased when HO-1 is knocked down by shRNA. In addition, we demonstrated carbon monoxide (CO) and p38MAPK are the cofactors in this signal pathway. In vivo animal model demonstrated HO-1 inhibited the tumor growth. In conclusion, in vitro and in vivo data show HO-1 inhibits the human HCC cells migration and tumor growth by suppressing the expression of IL-6. The heme degradation product CO is a cofactor in this process and inhibits p38MAPK phosphorylation.

Pregnane X receptor suppresses proliferation and tumourigenicity of colon cancer cells.

BACKGROUND: Pregnane X receptor (PXR) is a nuclear receptor that regulates the metabolism and disposition of various xenobiotics and endobioitics. We investigated a novel PXR function in regulating colon tumourigenesis in this study. METHODS: Histochemistry, transfection, cell proliferation assay, anchorage-alpha-dependent assay, xenograft, immunohistochemistry, immunofluorescence flow cytometry. RESULTS: Using histochemistry analysis, we found that PXR expressions were lost or greatly diminished in many colon tumours. Ectopic expression of human PXR through stable transfection of PXR into colon cancer cell line HT29 significantly inhibited cell proliferation as determined by cell proliferation assay and anchorage-independent assay. Pregnane X receptor suppressed significantly HT29 xenograft tumour growth in nude mice compared with control (310+/-6.2 vs 120+/-6 mg, P<0.01). Immunohistochemistry and immunofluorescence analysis of Ki-67 on excised xenograft tumour tissues showed that PXR inhibited cancer cell proliferation. Furthermore, expressions of PXR and Ki-67 were mutually exclusive. The flow cytometry analysis indicated that PXR caused G(0)/G(1) cell-cycle arrest. p21(WAF1/CIP1) expression was markedly elevated whereas E2F1 expression was inhibited by PXR. CONCLUSION: PXR inhibits the proliferation and tumourigenicity of colon cancer cells by controlling cell cycle at G(0)/G(1) cell phase by regulating p21(WAF1/CIP1) and E2F/Rb pathways.

Randomised clinical trial: faecal microbiota transplantation for recurrent Clostridum difficile infection - fresh, or frozen, or lyophilised microbiota from a small pool of healthy donors delivered by colonoscopy.

BACKGROUND: Faecal microbiota transplantation (FMT) has become routine in managing recurrent C. difficile infection (CDI) refractory to antibiotics. AIM: To compare clinical response and improvements in colonic microbiota diversity in subjects with recurrent CDI using different donor product. METHODS: Seventy-two subjects with ≥3 bouts of CDI were randomised in a double-blind study to receive fresh, frozen or lyophilised FMT product via colonoscopy from 50 g of stool per treatment from eight healthy donors. Recipients provided stools pre- and 7, 14 and 30 days post-FMT for C. difficile toxin and, in a subset, microbiome composition by 16S rRNA gene profiling. RESULTS: Overall resolution of CDI was 87% during 2 months of follow-up after FMT. Stool samples before FMT had significantly decreased bacterial diversity with a high proportion of Proteobacteria compared to donors. Cure rates were highest for the group receiving fresh product seen in 25/25 (100%), lowest for the lyophilised product 16/23 (78%; P = 0.022 vs. fresh and 0.255 vs. frozen) and intermediate for frozen product 20/24 (P = 0.233 vs. fresh). Microbial diversity was reconstituted by day 7 in the subjects receiving fresh or frozen product. Improvement in diversity was seen by day 7 in those randomised to lyophilised material with reconstitution by 30 days. CONCLUSIONS: Comparative efficacy in faecal microbiota transplantation was observed in subjects receiving fresh or frozen faecal product from the same donors. The lyophilised product had a slightly lowered efficacy compared with fresh product, but it resembled other treatments in microbial restoration 1 month after faecal microbiota transplantation.

Spin-spin interaction in ethanolamine deaminase.

The adenosylcobalamin coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium catalyzes the deamination of aminoethanol to acetaldehyde and ammonia. The radical intermediate observed during steady state turnover of substrate aminoethanol has been characterized by continuous wave electron paramagnetic resonance (EPR) spectroscopy [J. Am. Chem. Soc. 121 (1999) 10522]. This study presents simulations of EPR spectra of this radical intermediate. Quantitative fits to the EPR spectra are achieved with a model of isotropic exchange and magnetic dipolar interaction between the substrate-derived radical and the Co(II) in the corrin ring. The simulated parameters are compared with those of substrate analog 2-aminopropanol-derived radical in the same enzyme. The comparison confirms that the aminoethanol-derived product radical interacts more weakly with the Co(II) than the 2-aminopropanol-derived radical and suggests that the reduction of isotropic exchange between the aminoethanol-derived product radical and the Co(II) is probably due to orientational-dependent wave function overlap. Successful fits to the radical line shapes of different isotope substitutions unequivocally establish that the observed radical intermediate is an pi-electron-based product radical. The derived principal hyperfine values for the 13C(alpha) and 1H(alpha) nucleus are consistent with previous electron nuclear double resonance (ENDOR) studies on similar radicals, thus providing reliable experimental hyperfine coupling constants for comparison with quantum mechanical-based calculations to gain further insight into the molecular structure of the observed radical.

Use of synthetic peptides and copolymers to study the substrate specificity and inhibition of the protein tyrosine kinase pp60c-src.

The ability of synthetic peptides and polypeptides to act as substrates and/or inhibitors of pp60c-src was examined. The random copolymer, poly(K4Y) had a threefold lower specificity than poly(E4Y). Peptides containing lysine vs. glutamate were also found to have a lower substrate specificity (Vmax:Km ratio). In order to assess the substrate specificity of acidic peptides, an assay protocol using DEAE-membranes was developed. Peptides containing a (YXE)5YXD motif (X = G, A, V, P, or norvaline) were tested as inhibitors and substrates of pp60c-src. The glycine-containing peptide was the best substrate having a specificity 16,000-fold higher than 5Val-angiotensin II, the most commonly used peptide substrate. Most of the peptides, except for the proline containing peptide, had Ki values of 20-100 microM. In a series of (XGE)5XGD peptides, where X = Y or F, tyrosine at position 10 was found to be the preferred site for accepting a phosphate. Analogs in which the glycine was replaced with alanine indicated that loss of flexibility around position 10 was detrimental to substrate specificity. Results suggest that conformational requirements of the peptides tested was important and substrate specificity was a more sensitive parameter than binding as measured by Ki values.

Tumor irradiation enhances the tumor-specific distribution of poly(L-glutamic acid)-conjugated paclitaxel and its antitumor efficacy.

The poly(L-glutamic acid)-paclitaxel (PG-TXL) conjugate has been shown to exhibit significantly greater antitumor activity than conventionally formulated paclitaxel (TXL) against solid tumors (Li et al., Cancer Res., 58: 2404-2409, 1998). Here we report that local tumor irradiation enhanced the distribution of PG-TXL given 24 h later to ovarian OCa-1 carcinoma implanted i.m. in C3Hf/Kam mice. Radiation significantly increased tumor uptake of PG-TXL and tumor vascular permeability, caused elevation of the serum concentration of vascular endothelial growth factor, and arrested OCa-1 cells in the G1 phase of cell cycle. The enhancement factors, as measured by incremental tumor growth delay compared with PG-TXL alone, ranged from 1.36-4.44. Complete tumor regression was also observed at a higher radiation dose (>10 Gy) and a higher PG-TXL dose (>80 mg equivalentTXL/kg). Furthermore, combined radiation and PG-TXL produced a significantly greater tumor growth delay than treatment with radiation and TXL when both drugs were given at the same equivalent TXL dose of 60 mg/kg 24 h after tumor irradiation (enhancement factors, 4.44 versus 1.50). These data suggest that conjugation of TXL to poly(L-glutamic acid) is necessary for improved response and that the supra-additive effect of combined radiation and PG-TXL therapy is due in part to modulation of the enhanced permeability and retention effect of macromolecules by radiation. We propose a treatment strategy combining radiation and macromolecular chemotherapy that may have important clinical implications in terms of scheduling and optimization of the therapeutic ratio.

Antitumor activity of poly(L-glutamic acid)-paclitaxel on syngeneic and xenografted tumors.

Poly(L-glutamic acid)-paclitaxel (PG-TXL) is a new water-soluble paclitaxel derivative that has shown remarkable antitumor activity against both ovarian and breast tumors. The purpose of this study was to test whether the antitumor efficacy of PG-TXL depends on tumor type, as is the case for paclitaxel, and to test whether paclitaxel-resistant tumors could be responsive to PG-TXL. We evaluated the therapeutic activity of PG-TXL against four syngeneic murine tumors (MCa-4, MCa-35, HCa-1, and FSa-II) inoculated i.m. into C3Hf/Kam mice, a human SKOV3ip1 ovarian tumor injected i.p. into nude mice, and a human MDA-MB-435Lung2 breast tumor grown in the mammary fat pad of nude mice. Two paclitaxel-responsive murine tumors, MCa-4 and MCa-35, showed significant growth delay with PG-TXL given as a single i.v. injection at its maximum tolerated dose of 160 mg of equivalent paclitaxel/kg or even at a lower dose of 120 mg of equivalent paclitaxel/kg. The other two murine tumors, HCa-1 and FSa-II, did not respond particularly well to either of the two agents, although significant growth delay was observed for both tumors with PG-TXL. In mice with SKOV3ip1 tumors, the median survival times for mice treated with PG alone and PG-TXL at doses of 60 or 120 mg of equivalent paclitaxel/kg were 43, 61, and 75 days, respectively; no survival difference was found between paclitaxel-treated and Cremophor vehicle-treated mice. In mice with MDA-MB-435Lung2 tumor, PG-TXL at a dose of 120 mg of equivalent paclitaxel/kg produced regression of the tumor in 50% of the animals, and in the remaining mice, micrometastases in the lung were found only in 25% of the animals. In comparison, treatment with paclitaxel at 60 mg/kg did not result in tumor regression, and the rate of lung metastases was 42%. These results clearly demonstrate that PG-TXL has significant therapeutic activity against breast and ovarian tumors tested in this study. Future studies to elucidate the mechanism of action of PG-TXL and to assess its clinical applications are warranted.

Microbial characteristics of a methanogenic phenol-degrading sludge.

Microbial properties of a methanogenic granular phenol-degrading sludge were characterized using the 16S rRNA/DNA-based techniques, including polymerase chain reaction (PCR) amplification, cloning, DNA sequencing, and fluorescence in situ hybridization (FISH). The sludge was sampled from an upflow anaerobic sludge blanket reactor, which removed 98% of phenol (up to 1260 mg/l) in wastewater at 26 degrees C with 12 hours of hydraulic retention. Based on DNA analysis, the Eubacteria in the sludge was composed of 13 operational taxonomy units (OTUs). Two OTUs, one resembling Clostridium and the other remotely resembling Desulfotomaculum, were likely responsible for the conversion of phenol to benzoate, which was further degraded by five Syntrophus-resembling OTUs to acetate and H2/CO2; methanogens lastly converted acetate and H2/CO2 into methane. The role of six remaining OTUs remains unclear. Overall, the sludge was composed of 26 +/- 6% Eubacteria and 74 +/- 9% methanogens, of which 54 +/- 6% were acetotrophic Methanosaetaceae, 14 +/- 3% and 3 +/- 2% were hydrogenotrophic Methanomicrobiales and Methanobacteriaceae, respectively.