The trimeric Hef-associated nuclease HAN is a 3'→5' exonuclease and is probably involved in DNA repair.

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN's function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3'-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3'-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.

The crystal structure of Pyrococcus furiosus RecJ implicates it as an ancestor of eukaryotic Cdc45.

RecJ nucleases specifically degrade single-stranded (ss) DNA in the 5' to 3' direction. Archaeal RecJ is different from bacterial RecJ in sequence, domain organization, and substrate specificity. The RecJ from archaea Pyrococcus furiosus (PfuRecJ) also hydrolyzes RNA strands in the 3' to 5' direction. Like eukaryotic Cdc45 protein, archaeal RecJ forms a complex with MCM helicase and GINS. Here, we report the crystal structures of PfuRecJ and the complex of PfuRecJ and two CMPs. PfuRecJ bind one or two divalent metal ions in its crystal structure. A channel consisting of several positively charged residues is identified in the complex structure, and might be responsible for binding substrate ssDNA and/or releasing single nucleotide products. The deletion of the complex interaction domain (CID) increases the values of kcat/Km of 5' exonuclease activity on ssDNA and 3' exonuclease activity on ssRNA by 5- and 4-fold, respectively, indicating that the CID functions as a regulator of enzymatic activity. The DHH domain of PfuRecJ interacts with the C-terminal beta-sheet domain of the GINS51 subunit in the tetrameric GINS complex. The relationship of archaeal and bacterial RecJs, as well as eukaryotic Cdc45, is discussed based on biochemical and structural results.

Thermococcus Eurythermalis Endonuclease IV Can Cleave Various Apurinic/Apyrimidinic Site Analogues in ssDNA and dsDNA.

Endonuclease IV (EndoIV) is a DNA damage-specific endonuclease that mainly hydrolyzes the phosphodiester bond located at 5' of an apurinic/apyrimidinic (AP) site in DNA. EndoIV also possesses 3'-exonuclease activity for removing 3'-blocking groups and normal nucleotides. Here, we report that Thermococcus eurythermalis EndoIV (TeuendoIV) shows AP endonuclease and 3'-exonuclease activities. The effect of AP site structures, positions and clustered patterns on the activity was characterized. The AP endonuclease activity of TeuendoIV can incise DNA 5' to various AP site analogues, including the alkane chain Spacer and polyethylene glycol Spacer. However, the short Spacer C2 strongly inhibits the AP endonuclease activity. The kinetic parameters also support its preference to various AP site analogues. In addition, the efficient cleavage at AP sites requires ≥2 normal nucleotides existing at the 5'-terminus. The 3'-exonuclease activity of TeuendoIV can remove one or more consecutive AP sites at the 3'-terminus. Mutations on the residues for substrate recognition show that binding AP site-containing or complementary strand plays a key role for the hydrolysis of phosphodiester bonds. Our results provide a comprehensive biochemical characterization of the cleavage/removal of AP site analogues and some insight for repairing AP sites in hyperthermophile cells.

pSW2, a Novel Low-Temperature-Inducible Gene Expression Vector Based on a Filamentous Phage of the Deep-Sea Bacterium Shewanella piezotolerans WP3.

A low-temperature-inducible protein expression vector (pSW2) based on a filamentous phage (SW1) of the deep-sea bacterium Shewanella piezotolerans WP3 was constructed. This vector replicated stably in Escherichia coli and Shewanella species, and its copy number increased at low temperatures. The pSW2 vector can be utilized as a complementation plasmid in WP3, and it can also be used for the production of complex cytochromes with multiple heme groups, which has the potential for application for metal ion recovery or bioremediation. Promoters of low-temperature-inducible genes in WP3 were fused into the vector to construct a series of vectors for enhancing protein expression at low temperature. The maximum green fluorescent protein intensity was obtained when the promoter for the hfq gene was used. The WP3/pSW2 system can efficiently produce a patatin-like protein (PLP) from a metagenomic library that tends to form inclusion bodies in E. coli. The yields of PLP in the soluble fraction were 8.3 mg/liter and 4.7 mg/liter of culture at 4°C and 20°C, respectively. Moreover, the pSW2 vector can be broadly utilized in other Shewanella species, such as S. oneidensis and S. psychrophila.

Bacterial and archaeal diversities in Yunnan and Tibetan hot springs, China.

Thousands of hot springs are located in the north-eastern part of the Yunnan-Tibet geothermal zone, which is one of the most active geothermal areas in the world. However, a comprehensive and detailed understanding of microbial diversity in these hot springs is still lacking. In this study, bacterial and archaeal diversities were investigated in 16 hot springs (pH 3.2-8.6; temperature 47-96°C) in Yunnan Province and Tibet, China by using a barcoded 16S rRNA gene-pyrosequencing approach. Aquificae, Proteobacteria, Firmicutes, Deinococcus-Thermus and Bacteroidetes comprised the large portion of the bacterial communities in acidic hot springs. Non-acidic hot springs harboured more and variable bacterial phyla than acidic springs. Desulfurococcales and unclassified Crenarchaeota were the dominated groups in archaeal populations from most of the non-acidic hot springs; whereas, the archaeal community structure in acidic hot springs was simpler and characterized by Sulfolobales and Thermoplasmata. The phylogenetic analyses showed that Aquificae and Crenarchaeota were predominant in the investigated springs and possessed many phylogenetic lineages that have never been detected in other hot springs in the world. Thus findings from this study significantly improve our understanding of microbial diversity in terrestrial hot springs.

Abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China.

It has been suggested that archaea carrying the accA gene, encoding the alpha subunit of the acetyl CoA carboxylase, autotrophically fix CO2 using the 3-hydroxypropionate/4-hydroxybutyrate pathway in low-temperature environments (e.g., soils, oceans). However, little new information has come to light regarding the occurrence of archaeal accA genes in high-temperature ecosystems. In this study, we investigated the abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China, using DNA- and RNA-based phylogenetic analyses and quantitative polymerase chain reaction. The results showed that archaeal accA genes were present and expressed in the investigated Yunnan hot springs with a wide range of temperatures (66-96 °C) and pH (4.3-9.0). The majority of the amplified archaeal accA gene sequences were affiliated with the ThAOA/HWCG III [thermophilic ammonia-oxidizing archaea (AOA)/hot water crenarchaeotic group III]. The archaeal accA gene abundance was very close to that of AOA amoA gene, encoding the alpha subunit of ammonia monooxygenase. These data suggest that AOA in terrestrial hot springs might acquire energy from ammonia oxidation coupled with CO2 fixation using the 3-hydroxypropionate/4-hydroxybutyrate pathway.

[Research on the method of copper converting process determination based on emission spectrum analysis].

A method of copper converting process determination based on PbO/PbS emission spectrum analysis was described. According to the known emission spectrum of gas molecules, the existence of PbO and PbS was confirmed in the measured spectrum. Through the field experiment it was determined that the main emission spectrum of the slag stage was from PbS, and the main emission spectrum of the copper stage was from PbO. The relative changes in PbO/PbS emission spectrum provide the method of copper converting process determination. Through using the relative intensity in PbO/PbS emission spectrum the copper smelting process can be divided into two different stages, i.e., the slag stage (S phase) and the copper stage (B phase). In a complete copper smelting cycle, a receiving telescope of appropriate view angle aiming at the converter flame, after noise filtering on the PbO/PbS emission spectrum, the process determination agrees with the actual production. Both the theory and experiment prove that the method of copper converting process determination based on emission spectrum analysis is feasible.

Fabrication of bacterial cellulose with TiO2-ZnO nanocomposites as a multifunctional membrane for water remediation.

Superhydrophilic/underwater superoleophobic (SUS) membrane technology has attracted extensive attention for water purification. However, the fabrication of multifunctional membranes to satisfy the complex wastewater treatment is still a big challenge. In this work, bacterial cellulose (BC) based multifunctional SUS membranes were designed for water purification. Membranes were prepared by blending BC nanofibers with TiO2 nanoparticles (NPs), and further modified by the in situ growth of ZnO-NPs. The composite membranes showed oil/water (o/w) separation under a small driving pressure (0.2-0.3 bar) with a flux rate of 8232.81 ± 212 L m-2h-1 and with a high separation efficiency (>99.9%). Membranes could also separate oil-in-water emulsion with a separation flux of 1498 ± 74 L m-2h-1 and with high efficiency (99.25%). Moreover, the composite membrane exhibited photocatalytic activity under visible light with a high efficiency (>92%). The composite membranes were also investigated for antibacterial activity against Gram-positive and Gram-negative bacterial strains. This work may inspire the fabrication of next-generation multifunctional membranes for wastewater treatment, particularly oily wastewater, dyes and microbial contaminated water.

Self-assembly of peptide nanofibers with chirality-encoded antimicrobial activity.

The nanostructured antimicrobial agents, self-assembled by the antimicrobial peptides (AMPs), represent an intriguing platform for the treatment of pathogens. Although the structural characteristics significantly influence antimicrobial functionality, the role of chirality is usually ignored and still unclear. Herein, two homochiral AMPs (all L- or all D-amino acids), including C16-LV4LR4 (LL) and C16-DV4DR4 (DD), and a heterochiral AMP with alternating D-/L-amino acids, C16-DV4LR4 (DL), were self-assembled into left-handed, right-handed, and right-handed helical nanofibers, respectively. The valine configuration determined the supramolecular chirality of the nanofibers. However, the DL molecules exhibited a highly aggregated propensity to form more stable helical nanofibers with a lower degree of twist and a larger helical pitch. This characteristic resulted in the optimal antimicrobial activity of the DL nanofibers against both Gram-negative and Gram-positive bacteria. Furthermore, the membrane permeability assay confirmed the higher activity for damaging the cell membrane by the DL nanofibers. These results demonstrated the significance of molecular chirality in directing the self-assembly of the amphiphilic peptides, eventually affecting their antimicrobial activity. This study opens up the possibility to fabricate promising nanostructured antimicrobial materials by controlling the chirality and structure of the materials.

In situ regulation of bacterial cellulose networks by starch from different sources or amylose/amylopectin content during fermentation.

Bacterial cellulose (BC) is a promising biopolymer, but its three-dimensional structure needs to be controllable to be used in multiple fields. BC has some advantages over other types of cellulose, not only in terms of purity and properties but also in terms of modification (in situ modification) during the synthesis process. Here, starches from different sources or with amylose/amylopectin content were added to the growth medium to regulate the structural properties of BC in-situ. The obtained BC membranes were further modified by superhydrophobic treatment for oil-water separation. Starches alter the viscosity of the medium, thus affecting bacterial motility and cellulose synthesis, and adhere to the microfibers, limiting their further polymerization and ultimately altering the membrane porosity, pore size, and mechanical properties perpendicular to the BC fibril layer direction. The average pore diameter of the BC/PS membrane increased by 1.94 times compared to the initial BC membrane. The chemically modified BC/PS membrane exhibited super-hydrophobicity (water contact angle 167°), high oil-water separation flux (dichloromethane, 23,205 Lm-2 h-1 MPa-1), high separation efficiency (>97%). The study provides a foundation for developing methods to regulate the network structure of BC and broaden its application.

New cytotoxic metabolites from a deep-sea-derived fungus, Phialocephala sp., strain FL30r.

Two new sorbicillinoids, 1 and 2, together with a novel benzofuranone derivative named phialofurone (3), were isolated from a deep-sea sediment-derived fungus, Phialocephala sp. Their structures were established on the basis of spectroscopic data. All compounds displayed cytotoxic effects against P388 (IC(50) values of 11.5±1.4, 0.1±0.1, and 0.2±0.01 µM, resp.) and K562 (IC(50) values of 22.9±0.8, 4.8±0.3 and 22.4±0.9 µM, resp.) cell lines.

[Significance of spinal sagittal balance for guiding acetabular prosthesis positioning].

Since the concept of "safe area" put forward by Lewinnek, it has been widely recognized. While in recent years, many scholars have found that even if the acetabular prosthesis was placed on the "safe area", there were still many unexplained dislocation after total hip arthroplasty. And scholars began to question whether the "safe area" is really suitable for all patients. Spinal degeneration, deformity, lumbar fusion, etc. will lead to spine sagittal imbalance and changes in pelvic activity, which could lead to changes in acetabular orientation, and ultimately lead to edge loading, wear, impact, and even dislocation after total hip replacement. From the perspective of wear, impact and dislocation, it is determined by the functional positioning of the acetabular cup, not the anatomical positioning. The anatomical positioning and functional positioning of the neutral pelvic acetabular cup in the standing position can be considered equivalent. For pelvic rotation more than 20°, functional placement needs to be considered. In recent years, as the understanding of the internal relationship between the spine-pelvis-hip joint has become more and more profound, some scholars further classify the hip-spine relationship according to whether the spine is stiff or deformed, and propose corresponding acetabulums according to different types of hip-spine relationships The function of placement, so as to achieve a stable artificial hip joint. Therefore, it is of great significance to fully assess whether the patient's sagittal plane is balanced before surgery to guide artificial hip replacement surgery.

Fabrication of Bacterial Cellulose-Based Dressings for Promoting Infected Wound Healing.

Bacterial cellulose (BC) holds several unique properties such as high water retention capability, flexibility, biocompatibility, and high absorption capacity. All these features make it a potential material for wound healing applications. However, it lacks antibacterial properties, which hampers its applications for infectious wound healings. This study reported BC-based dressings containing ε-polylysine (ε-PL), cross-linked by a biocompatible and mussel-inspired polydopamine (PDA) for promoting infectious wound healing. BC membranes were coated with PDA by a simple self-polymerization process, followed by treating with different contents of ε-PL. The resulted membranes showed strong antibacterial properties against tested bacteria by both in vitro and in vivo evaluations. The membranes also exhibited hemocompatibility and cytocompatibility by in vitro investigations. Moreover, the functionalized membranes promoted infected wound healing using Sprague-Dawley rats as a model animal. A complete wound healing was observed in the group treated with functionalized membranes, while wounds were still open for control and pure BC groups in the same duration. Histological investigations indicated that the thickness of newborn skin was greater and smoother in the groups treated with modified membranes in comparison to neat BC or control groups. These results revealed that the functionalized membranes have great potential as a dressing material for infected wounds in future clinical applications.

Sustainable, superhydrophobic membranes based on bacterial cellulose for gravity-driven oil/water separation.

Bacterial cellulose (BC) is a substrate material with high purity and robust mechanical strength, but due to its small pore size and relatively expensive price, it is restricted as an oil-/water separation membrane. In this study, cheaper plant cellulose needle-leaf bleached kraft pulp (NBKP) was added to BC to increase the pore size of the composite membrane, and a superhydrophobic/superoleophilic membrane was prepared for oil-/water separation. The modified membrane surface displayed a petal-like micro-structure and a water contact angle (WCA) of 162.3°, while the oil contact angle was decreased to 0°. What's more, the membrane exhibited excellent oil-/water separation under gravity, recyclability, and a separation efficiency (>95 %), and it was both pH and salt resistant. The membrane also remained durably hydrophobic after 10 separation cycles. And the separation methodology is expected to be highly energy-efficient.

Ginsenoside Rg1 modulation on thrombospondin-1 and vascular endothelial growth factor expression in early renal fibrogenesis in unilateral obstruction.

Renal interstitial fibrosis is the major histopathological change seen in a variety of renal disorders and is closely related to renal dysfunction. Progressive interstitial fibrosis accompanied by the loss of renal tubules and interstitial capillaries typifies all progressive renal disease. Thrombospondin-1 (TSP-1) is a major angiogenic inhibitor. It is demonstrated that TSP-1 levels were correlated with the loss of glomerular and peritubular capillaries and TSP-1 could promote renal scarring by effects on the endothelium. It has been reported that ginsenoside Rg1 inhibited renal interstitial fibrosis in rats via suppressing the expression of TSP-1. The present study was designed to examine whether ginsenoside Rg1 could modulate the integrity of the microvasculature and hence affect the progression of renal fibrosis in a rat unilateral ureteral obstruction (UUO) model. In UUO control kidneys, associated with interstitial fibrosis, lower peritubular capillary densities were prominent. These changes were all improved by ginsenoside Rg1 treatment. Interestingly, ginsenoside Rg1 decreased the expression of TSP-1 and enhanced vascular endothelial growth factor (VEGF) expression. The results show for the first time that ginsenoside Rg1 can evidently inhibit renal interstitial fibrosis in rats with UUO. The mechanism might be related to suppression of the expression of TSP-1 and to repair of the peritubular capillary.

A new cytotoxic metabolite from a deep sea derived fungus, Phialocephala sp.

A new sesquiterpene hydroquinone (1) was isolated from a deep sea sediment derived fungus, Phialocephala sp.. Its structure and stereochemistry were established on the basis of spectroscopic data and optical rotation. This compound was tested for cytotoxicity against P388 (murine leukemia cell) and K562 (human leukemia cell) cell lines, and displayed strong cytotoxic effects with IC50 value of 0.16 and 0.05 micromol x L(-1), separately.

[Leukemia inhibitory factor suppresses renal interstitial fibroblast activation induced by transforming growth factor].

OBJECTIVE: To investigate the effects of leukemia inhibitory factor (LIF) on renal interstitial fibroblast activation following induction by transforming growth factor beta 1 (TGF-beta1). METHODS: Normal rat interstitial fibroblast cells (NRK/49F) were treated with TGF-beta1 and TGF-beta1, combining with LIF respectively for different duration with different concentration. Changes in cell morphology and expression of alpha-SMA were evaluated with electronic microscope and Western blot respectively. The collagen I in the supernatant was detected with ABC-ELISA. RESULTS: TGF-beta1 induced renal interstitial fibroblast activation, and this was accompanied by significant morphological transformations and secretion of collagen I. Co-culturing of cells with LIF blocked the morphological transformation. In addition, LIF inhibited TGF-beta1-induced expression of alpha-SMA mRNA and protein (P < 0.01), and decreased the levels of collagen I (P < 0.01) in a dose-dependent manner. CONCLUSION: LIF suppresses TGF-beta1-induced activation and collagen I secretion of cultured renal interstitial fibroblasts.

[A method for resolving spectra shift in the urban air quality monitoring system (DOAS)].

In the urban air quality monitoring system, there is spectra shift which is caused by environment factors on the optical part (temperature and optic fiber position), or by the self-change of Xe-lamp. Relative spectra shift will occur if the shift of lamp-spectrum and air-spectrum is inconsistent which has direct influences on the accuracy of the measurement results. So the match of wavelength between lamp-spectrum and air-spectrum should be considered when we retrieve pollutants concentration measurement of trace gas in the atmosphere through DOAS method. Based on the study of the unique structures for Xe-lamp emitting spectrum, a method for the calibration of two signal spectra using Xe-lamp emitting peak and least square fitting is given. The results show that, the impact of spectrum shift can be reduced by this method for retrieving results.

Reusable ternary PVA films containing bacterial cellulose fibers and ε-polylysine with improved mechanical and antibacterial properties.

The combination of high mechanical properties, antibacterial activity and a green synthesis of the polyvinyl alcohol (PVA) based films remains challenging. This study presents a ternary system of PVA films containing bacterial cellulose (BC) and epsilon-polylysine (ε-PL) by a green solution casting method. The prepared composite films showed more than 99% antibacterial properties against both Staphylococcus aureus and Escherichia coli bacteria. Moreover, the films were collected after a single use and were reused twice, which still exhibited strong antibacterial activity. The films showed thermal stability and higher mechanical properties as compared to pure PVA films. In addition, the cytotoxicity of the films was evaluated by MTT assay against NIH 3T3 mouse fibroblast cells. The results showed no toxicity of the films towards tested cells. We believe that these antibacterial films may find applications in active food packaging and biomedical fields.