Minimally invasive surgery for hilar cholangiocarcinoma: a multicenter retrospective analysis of 158 patients.

BACKGROUND: Curative resection of hilar cholangiocarcinoma (HC) is typically carried out using open surgery. In the present study, we examined the safety (postoperative complication) and effectiveness (resection margin status and patient survival) of minimally invasive surgery (MIS) for HC. METHODS: This retrospective analysis included 158 patients receiving MIS for HC at 10 participating centers between December 2013 and November 2019. Patient demographics, surgical outcomes, and oncological outcomes were retrospectively analyzed. RESULTS: Clinical information obtained from 10 different clinical centers did not show any evident cohort-bias clustering. One hundred and twenty-six (79.7%) patients underwent LRHC, 12 (7.6%) patients underwent RARHC, conversion to an open procedure occurred in 20 (12.7%) patients. The operation time and estimated blood loss were 410.8 ± 128.9 min and 477.8 ± 706.3 mL, respectively. The surgical radicality of the 158 patients was R0, 129 (81.6%); R1, 20 (18.4%) and R2, 9 (5.7%). Grades I-II complications was occurred in 68 (43.0%) patients. Severe morbidity (grade III-V) occurred in 14 (8.7%) patients. The median overall survival in whole cohort was 25.4 months. The overall survival rate was 67.6% at year 1, 28.8% at year 3, and 19.2% at year 5. Comparing the first half of MISHC performed by each center with the following cases, the operation time and postoperative hospital stay does not decrease with the increasing cases. On literature review, MISHC is non-inferior to open surgery at least in perioperative period. CONCLUSIONS: In this Chinese MIS for HC multicenter study, the largest to date, long-term overall survival rates after MIS appear comparable to those reported in current open series. Further randomized controlled trials are necessary to assess the global impact of MISHC.

Substantial Epigenetic Variation Causing Flower Color Chimerism in the Ornamental Tree Prunus mume Revealed by Single Base Resolution Methylome Detection and Transcriptome Sequencing.

Epigenetic changes caused by methylcytosine modification participate in gene regulation and transposable element (TE) repression, resulting in phenotypic variation. Although the effects of DNA methylation and TE repression on flower, fruit, seed coat, and leaf pigmentation have been investigated, little is known about the relationship between methylation and flower color chimerism. In this study, we used a comparative methylomic⁻transcriptomic approach to explore the molecular mechanism responsible for chimeric flowers in Prunus mume "Danban Tiaozhi". High-performance liquid chromatography-electrospray ionization mass spectrometry revealed that the variation in white (WT) and red (RT) petal tissues in this species is directly due to the accumulation of anthocyanins, i.e., cyanidin 3,5-O-diglucoside, cyanidin 3-O-glucoside, and peonidin 3-O-glucoside. We next mapped the first-ever generated methylomes of P. mume, and found that 11.29⁻14.83% of the genomic cytosine sites were methylated. We also determined that gene expression was negatively correlated with methylcytosine level in general, and uncovered significant epigenetic variation between WT and RT. Furthermore, we detected differentially methylated regions (DMRs) and DMR-related genes between WT and RT, and concluded that many of these genes, including differentially expressed genes (DEGs) and transcription factor genes, are critical participants in the anthocyanin regulatory pathway. Importantly, some of the associated DEGs harbored TE insertions that were also modified by methylcytosine. The above evidence suggest that flower color chimerism in P. mume is induced by the DNA methylation of critical genes and TEs.

Distribution of 45S rDNA in Modern Rose Cultivars (Rosa hybrida), Rosa rugosa, and Their Interspecific Hybrids Revealed by Fluorescence in situ Hybridization.

To elucidate the evolutionary dynamics of the location and number of rDNA loci in the process of polyploidization in the genus Rosa, we examined 45S rDNA sites in the chromosomes of 6 modern rose cultivars (R. hybrida), 5 R. rugosa cultivars, and 20 hybrid progenies by fluorescence in situ hybridization. Variation in the number of rDNA sites in parents and their interspecific hybrids was detected. As expected, 4 rDNA sites were observed in the genomes of 4 modern rose cultivars, while 3 hybridization sites were observed in the 2 others. Two expected rDNA sites were found in 2 R. rugosa cultivars, while in the other 3 R. rugosa cultivars 4 sites were present. Among the 20 R. hybrida × R. rugosa offspring, 13 carried the expected number of rDNA sites, and 1 had 6 hybridization sites, which exceeded the expected number by far. The other 6 offspring had either 2 or 3 hybridization sites, which was less than expected. Differences in the number of rDNA loci were observed in interspecific offspring, indicating that rDNA loci exhibit instability after distant hybridization events. Abnormal chromosome pairing may be the main factor explaining the variation in rDNA sites during polyploidization.

Thermodynamic signatures of the antigen binding site of mAb 447-52D targeting the third variable region of HIV-1 gp120.

The third variable region (V3) of HIV-1 gp120 plays a key role in viral entry into host cells; thus, it is a potential target for vaccine design. Human monoclonal antibody (mAb) 447-52D is one of the most broadly and potently neutralizing anti-V3 mAbs. We further characterized the 447-52D epitope by determining a high-resolution crystal structure of the Fab fragment in complex with a cyclic V3 and interrogated the antigen-antibody interaction by a combination of site-specific mutagenesis, isothermal titration calorimetry (ITC) and neutralization assays. We found that 447-52D's neutralization capability is correlated with its binding affinity and at 25 °C the Gibbs free binding energy is composed of a large enthalpic component and a small favorable entropic component. The large enthalpic contribution is due to (i) an extensive hydrogen bond network, (ii) a π-cation sandwiching the V3 crown apex residue Arg(315), and (iii) a salt bridge between the 447-52D heavy chain residue Asp(H95) and Arg(315). Arg(315) is often harbored by clade B viruses; thus, our data explained why 447-52D preferentially neutralizes clade B viruses. Interrogation of the thermodynamic signatures of residues at the antigen binding interface gives key insights into their contributions in the antigen-antibody interaction.

Proximal glycans outside of the epitopes regulate the presentation of HIV-1 envelope gp120 helper epitopes.

Glycosylation of HIV-1 envelope gp120 determines not only the proper structure, but also the immune responses against this Ag. Although glycans may be part of specific epitopes or shield other epitopes from T cells and Abs, this study provides evidence for a different immunomodulatory function of glycans associated with gp120 residues N230 and N448. These glycans are required for efficient MHC class II-restricted presentation of nearby CD4 T cell epitopes, even though they are not part of the epitopes. The glycans do not affect CD4 T cell recognition of more distant epitopes and are not essential for the proper folding and function of gp120. Data on CD4 T cell recognition of N448 mutants combined with proteolysis analyses and surface electrostatic potential calculation around residue N448 support the notion that N448 glycan near the epitope's C terminus renders the site to be surface accessible and allows its efficient processing. In contrast, the N230 glycan contributes to the nearby epitope presentation at a step other than the proteolytic processing of the epitope. Hence, N-glycans can determine CD4 T cell recognition of nearby gp120 epitopes by regulating the different steps in the MHC class II processing and presentation pathway after APCs acquire the intact gp120 Ag exogenously. Modifications of amino acids bearing glycans at the C termini of gp120 helper epitopes may prove to be a useful strategy for enhancing the immunogenicity of HIV-1 envelope gp120.

The complete chloroplast genome of ornamental plant Ruellia simplex C.Wright (Acanthaceae).

Ruellia simplex C.Wright is a perennial plant of the Acanthaceae, which has significant ornamental value. Because of its strong adaptability, it is widely planted in Chinese rural areas. Based on sequencing data from Illumina, the first complete chloroplast (cp) genome of Ruellia simplex C.Wright is reported in this paper. This cp genome was 143,016bp in length, including a large single-copy region (LSC) of 91,857bp, a small single-copy (SSC) of 17,591bp and two inverted repeat regions (IRs) of 16,784bp. It contained 128 genes, 35 transfer RNA genes, 8 ribosomal RNA genes, with an overall GC content of 38.41%. Additionally, the phylogenetic analysis showed that Ruellia simplex is closely related to Strobilanthes cusia (NC_037485), Strobilanthes bantonensis (MT576695) and Echinacanthus attenuatus (NC_039762). The results of this study provide valuable information for the continued study of its species evolution, genetic engineering and germplasm resource utilization.

Treatment of osteomyelitis by liposomal gentamicin-impregnated calcium sulfate.

OBJECTIVES: Traditional therapy of staphylococcal osteomyelitis is ineffective in producing complete sterilization of infected bones due to the formation of the Staphylococcus aureus biofilms. The aim of this study was to develop a new drug-delivery system of antibiotics for treatment of chronic experimental osteomyelitis. METHODS: In the current work, cationic liposomal gentamicin was prepared and impregnated in calcium sulfate (CS), and tested for anti-biofilm activities in vitro and in vivo. RESULTS AND CONCLUSIONS: The combination of liposomal gentamicin and CS showed initial burst-release of active liposomal gentamicin and had continuous-release (12 days). Liposomal gentamicin released from CS had the same anti-biofilm activity with the liposomal gentamicin prepared freshly. Meanwhile, both agents were more effective relative to free gentamicin at low drug concentration. Therapeutic trials with antibiotics given intravenously revealed that free gentamicin for 14 days was ineffective in sterilizing bone. Treatment with liposomal gentamicin for 14 days resulted in recovery of 33.3% of treated animals, which was the lower slightly than the result treated with implantation of gentamicin-impregnated CS (66.7%). Complete sterilization of bone tissues on cultures (100% cure) was obtained only in the group of liposomal gentamicin-impregnated CS treated for 14 days. The new drug-delivery system was effective in preventing biofilm infection in a contaminated defect, and it could also be used clinically for bacterial infections in the conditions like plaque formation or in arresting biofilm formation in the implanted devices or dead bone of osteomyelitis.

Author Correction: The rise and fall of the Old World savannah fauna and the origins of the African savannah biome.

In the version of this Article originally published, each of the five panels in Fig. 5 incorrectly contained a black diagonal line across the plot. This has now been corrected.

The rise and fall of the Old World savannah fauna and the origins of the African savannah biome.

Despite much interest in the ecology and origins of the extensive grassland ecosystems of the modern world, the biogeographic relationships of savannah palaeobiomes of Africa, India and mainland Eurasia have remained unclear. Here we assemble the most recent data from the Neogene mammal fossil record in order to map the biogeographic development of Old World mammalian faunas in relation to palaeoenvironmental conditions. Using genus-level faunal similarity and mean ordinated hypsodonty in combination with palaeoclimate modelling, we show that savannah faunas developed as a spatially and temporally connected entity that we term the Old World savannah palaeobiome. The Old World savannah palaeobiome flourished under the influence of middle and late Miocene global cooling and aridification, which resulted in the spread of open habitats across vast continental areas. This extensive biome fragmented into Eurasian and African branches due to increased aridification in North Africa and Arabia during the late Miocene. Its Eurasian branches had mostly disappeared by the end of the Miocene, but the African branch survived and eventually contributed to the development of Plio-Pleistocene African savannah faunas, including their early hominins. The modern African savannah fauna is thus a continuation of the extensive Old World savannah palaeobiome.

Some properties of the interaction between 2,2'-diselenadibenzoic acid and serum albumins.

The binding of 2,2'-diselenadibenzoic acid to bovine serum albumin (BSA) and human serum albumin (HSA) was studied by using fluorescence spectroscopy. The measurement was performed in Tris-HCl buffer aqueous medium at pH = 7.40. The quenching constant at 303 K was (3.277 +/- 0.046) x 10(13) L mol(-1) s(-1) for BSA, and (3.946 +/- 0.002) x 10(12) L mol(-1) s(-1) for HSA. Decreased quenching was observed in association with increased temperature. Our findings show that the observed binding constant is dependent on the ionic strength of the medium. It is said that electrostatic interactions play a role in the binding of 2,2'-diselenadibenzoic acid to serum albumin, in addition to the hydrophobic association. The decrease of the linearity of S-V plot demonstrates reduced binding of ligand to the protein in the presence of anionic surfactants such as sodium dodecyl sulfate (SDS), which indicates that 2,2'-diselenadibenzoic acid most likely binds to the hydrophobic pockets within sub-domain IIA of serum albumin, the same site as SDS.

Interaction of C17orf25 with ADP-ribose pyrophosphatase NUDT9 detected via yeast two-hybrid method.

The gene C17orf25 was isolated from the liver by RACE PCR. nudt9 gene was screened by yeast two-hybrid method in MatchMaker human HeLa cDNA library. NUDT9 is an enzyme that has pyrophosphatase activity with ADP-ribose as its substrate. Fusion expression of C17orf25 and GFP and computer analysis showed that C17orf25 was probably located in mitochondria. Furthermore, C17orf25 may suppress the cell growth by interaction with NUDT9.

An Expression Release System for Heterogeneous Proteins in E.coli Mediated by the kil Gene.

An expression release system for heterogeneous proteins in Escherichia coli mediated by the colicin release gene(kil gene) was constructed. The system is based on the ability of the Kil protein to release periplasmic proteins into the growth medium. beta-lactamase, an E.coli periplasmic protein, and prolyl endopeptidase (PEP), a periplasmic protein of Aeromonas punctata subsp. punctata were used as report proteins. Commonly, these two proteins are seldom released into the growth medium. The results indicated that the released amounts of the beta-lactamase and of the prolyl endopeptidase were nearly 5 fold and 4 fold than control, respectively.

The Thioredoxin Reductase-deficient E.coli Mutant Enhances Expression into Solution of Recombinant Proteins Containing Cys Residues.

A 3D artificial protein, a salmon calcitonin hexa-polymer, a salmon calcitonin octo-polymer and a human prourokinase, was expressed in the cytoplasma of E.coli GJ980(trxB(-)) mutant. These recombinant proteins containedcysteine residues of different length ranging from 12-22 residues. The mutation was mapped to the gene for thioredoxin reductase(trxB) and was found to eliminate the activity of this enzyme, which was thought to contribute to the sulfhydryl reducing potential of the cytoplasm. Recombinant salmon calcitonin hexapolymer, salmon calcitonin octo-polymer and human prourokinase had more soluble form in cytoplasm of GJ980 mutants than in wild-type strain, while 3D-protein, which has nocysteine residue, still remain in insoluble form. Results indicate the GJ980(trxB(-)) strain allowed the formation of disulphide bonds in the cell cytoplasm which is believed to encourage correct folding and soluble expression of the recombinant proteins.

Cloning and expression of adiponectin and its globular domain, and measurement of the biological activity in vivo.

3T3-L1-adipocytes produce the adipocyte complement related protein of 30 kD (ACRP30), which is exclusively expressed in differentiated adipocytes. Decreased expression of ACRP30 correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin, the human homologue of ACRP30, circulates in human plasma at high levels. Plasma adiponectin levels have been reported to be decreased in some insulin-resistant states, such as obesity and type II diabetes mellitus. Here, full-length adiponectin and its C-terminal globular head domain (gAdiponectin) were expressed in Escherichia coli and gAdiponectin was used to immunize a rabbit to obtain polyclonal antiserum with titer of 10,000. Adiponectin was detected in human plasma with the use of gAdiponectin anti-serum by Western blot analysis, which was also detected by gACRP30 anti-serum. Injection in alloxan-treated rats with purified recombinant fusion adiponectin or gAdiponectin transiently abolished hyperglycemia. So adiponectin and gAdiponectin might have activity as a glucose lowering agent and potentially as a therapeutic for metabolic disease. All these results suggested that the recombinant protein had biological activity, and provided a useful tool in further studies.

Construction of a Novel Engineering Strain E.coli G830,which is Adoptable to High-cell-density Fermentation.

A novel engineered strain G830 adoptable to high-cell-density fermentation by integrating bacterial hemoglobin vhb (Vitreoscilla hemoglobin gene) into thr operon in the chromosome of PA1 blocking Pta-Ack metabolic pathway through the homologous recombination between the homologous fragments of integrated plasmid and that of chromosome. The engineered strain G830 was characterized by phenotype observation, PCR, thr mutant, acetate acid detection, Western blotting and VHb activity assays. In high dentity fermentation, the cellular respiration, energy metabolism, highest bacterial density and dry bacteria weight of the G830 strain were markedly better than control strains PA1 and BL21. The expression of recombinant prolyl endopeptidase (PEP) in G830 and PA1 under the above condition was high and stable. Their growth situation and fermentation parameters were similar with their parental strains without plasmids, and resided plasmids maintained stably in those strains. It revealed that the integral VHb and acetate metabolism pathway (Pta-Ack) block improved the growth of host strain under low-dissolved-oxygen conditions, enhanced the recombinant proteins production and reduced the accumulation of acetate harmful to bacterial growth. In conclusion, the novel engineering strain G830 was adoptable to high cell density fermentation.

Reaper homologue IBM1 in silkworm Bombyx mori induces apoptosis upon baculovirus infection.

In lieu of an adaptive immune system, apoptosis plays a central role in regulating cellular or environmental stimuli in Lepidopteran insect cells during viral infection. Bombyx mori IBM1 gene, a Drosophila Reaper orthologue, is localised in mitochondria and can induce a mitochondrial membrane potential loss. Further, expression of IBM1 is up-regulated rapidly to protect insect cells from viral infection. Ours is the first evidence to indicate that IBM1 could interact with BmNPV IAP2 following B. mori nucleopolyhedrosisvirus (BmNPV) infection. Our data indicate that IBM1 function might be reduced by viral anti-apoptotic genes when BmN cells are infected by BmNPV.

Exploring visual and motion saliency for automatic video object extraction.

This paper presents a saliency-based video object extraction (VOE) framework. The proposed framework aims to automatically extract foreground objects of interest without any user interaction or the use of any training data (i.e., not limited to any particular type of object). To separate foreground and background regions within and across video frames, the proposed method utilizes visual and motion saliency information extracted from the input video. A conditional random field is applied to effectively combine the saliency induced features, which allows us to deal with unknown pose and scale variations of the foreground object (and its articulated parts). Based on the ability to preserve both spatial continuity and temporal consistency in the proposed VOE framework, experiments on a variety of videos verify that our method is able to produce quantitatively and qualitatively satisfactory VOE results.

Development of microsatellite markers for Lagerstroemia indica (Lythraceae) and related species.

PREMISE OF THE STUDY: Microsatellite markers were developed and characterized to analyze genetic diversity within Lagerstroemia cultivars and related species. • METHODS AND RESULTS: Using simple sequence repeat (SSR)-enriched libraries, 11 species-specific polymorphic genomic SSRs were developed from L. indica 'Hong Die Fei Wu'. All primers were tested on 48 L. indica individuals from China, the United States, and France. The primers amplified four to 12 alleles per locus, including di-, tri-, and tetranucleotide repeats. Observed and expected heterozygosities ranged from 0.1875 to 0.7609 and 0.2836 to 0.8385, respectively. The primers were also highly cross-transferrable to L. subcostata, L. limii, L. fauriei, L. caudata, and L. speciosa. • CONCLUSIONS: The new primers will enlarge the bank of SSRs available to genetic research of Lagerstroemia. These SSR markers will facilitate population genetics and molecular marker-assisted selection of L. indica.

[Mechanism underlying the inhibitory effects of peroxisome proliferator-activated receptor γ agonists on transforming growth factor ß1 in adult skin fibroblasts].

OBJECTIVE: To study the mechanism underlying the inhibitory effects of peroxisome proliferator-activated receptor γ (PPARγ) agonists on transforming growth factor ß1 (TGF-ß(1))-induced scarring of skin. METHODS: Fibroblasts isolated from healthy adult skin were cultured in vitro and divided into blank control group (serum-free DMEM culture), TGF-ß(1) group (with stimulation of 10 ng/mL TGF-ß(1) for 48 hours), troglitazone group (with the same treatment as in TGF-ß(1) group after stimulation of 10 µmol/L troglitazone for 2 hours), and 15-dioxygen prostaglandin J2 (15d-PGJ2) group (with the same treatment as in TGF-ß(1) group after stimulation of 10 µmol/L 15d-PGJ2 for 2 hours) according to the stimulation added into DMEM. The expression of connective tissue growth factor (CTGF) was determined with Western blot. The mRNA levels of CTGF, matrix metalloproteinase-1 (MMP-1) and platelet-derived growth factor (PDGF) were determined with real-time fluorescence RT-PCR. Data were processed with one-way analysis of variance. RESULTS: The expression of CTGF at mRNA and protein levels in skin fibroblasts were significantly increased in TGF-ß(1) group as compared with control group; while expression of CTGF at mRNA and protein levels in 15d-PGJ2 and troglitazone groups were significantly decreased as compared with that in TGF-ß(1) group. The mRNA level of MMP-1 in TGF-ß(1) group (0.193 ± 0.051) was obviously lower than that in blank control group (1.281 ± 0.195, F = 12.811, P < 0.01), while the mRNA levels of MMP-1 in troglitazone group (0.417 ± 0.043) and 15d-PGJ2 group (0.485 ± 0.027) were significantly increased as compared with that in TGF-ß(1) group (F = 12.811, P values all below 0.01). The mRNA level of PDGF in TGF-ß(1) group (1.044 ± 0.237) was obviously higher than that in control group (0.349 ± 0.057, F = 16.848, P < 0.01), while the levels in troglitazone group (0.677 ± 0.055) and 15d-PGJ2 group (0.511 ± 0.017) were significantly decreased as compared with that in TGF-ß(1) group (F = 16.848, P values all below 0.01). CONCLUSIONS: The inhibitory effect of activated PPARγ on the expression of CTGF induced by TGF-ß(1) may be the main mechanism of its inhibitory effect on TGF-ß(1)-induced scarring on skin, and its influence on MMP-1 and PDGF may also be one of the underlying mechanisms.