Efficacy and safety analysis of hypofractionated and conventional fractionated radiotherapy in postoperative breast cancer patients.

OBJECTIVES: In this meta-analysis, we conducted a comparative analysis of the safety and efficacy of hypofractionated and conventional fractionated radiotherapy in individuals who had undergone surgery for breast cancer. METHODS: This study involved a systematic and independent review of relevant research articles published in reputable databases such as PubMed, Embase, Cochrane Library, and Web of Science. Two investigators conducted the review, which included studies published up to January 3, 2023. The quality of the eligible studies was evaluated and data were extracted using Review Manager software 5.4 (RevMan 5.4) to calculate odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: The analysis comprised 35 studies and encompassed a collective sample of 18,246 individuals diagnosed with breast cancer. We did not find a statistically significant disparity in efficacy between conventional fractionated (CF) radiotherapy and hypofractionated (HF) radiotherapy regarding local recurrence (LR; OR = 0.91, 95% CI: 0.76-1.09, P = 0.30), disease-free survival (DFS; OR = 1.20, 95% CI: 1.01-1.42, P = 0.03), and overall survival (OS; OR = 1.08, 95% CI: 0.93-1.26, P = 0.28). Concerning safety, there was no significant difference between the HF and CF regimens in terms of breast pain, breast atrophy, lymphedema, pneumonia, pulmonary fibrosis, telangiectasia, and cardiotoxicity. However, the HF regimen resulted in lower skin toxicity (OR = 0.43, 95% CI: 0.33-0.55, P < 0.01) and improved patient fatigue outcomes (OR = 0.73, 95% CI: 0.60 - 0.88, P < 0.01). CONCLUSIONS: Although there is no substantial difference in LR, DFS, OS, or many other side effects between the HF and CF regimens, the HF regimen reduces skin toxicity and relieves patient fatigue. If these two issues need to be addressed in clinical situations, the HF regimen may be a superior alternative to conventional radiotherapy in postoperative breast cancer patients.

Cardiorespiratory dose comparison among six radiotherapy regimens for patients with left-sided breast cancer.

There is uncertainty regarding the benefits and drawbacks of various radiation protocols for the treatment of left-sided breast cancer. To address this issue, we conducted a Bayesian network analysis. First, we searched several electronic databases for eligible literature. Next, we pooled the data from twelve studies concerning three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), and volumetric modulated arc therapy (VMAT), combined with either deep inspiratory breath-holding (DIBH) or free-breathing (FB) modalities. The integrated cardiac and pulmonary dosimetric indexes for all included treatments were compared using Bayesian networks. A direct meta-analysis indicated that for the two methods of 3D-CRT and IMRT, DIBH technology was more effective than FB in reducing the radiation dose to the heart and lungs. Additionally, according to the network results, DIBH was superior to FB in all six treatment options, regardless of whether the plan was 3D-CRT, IMRT, or VMAT. Besides, the combined data indicated that the FB-3D-CRT regimen had the weakest dosimetric advantage of all the treatments. Excluding FB-3D-CRT, each of the other five treatments had its own specific benefits. This is the first Bayesian study of several radiotherapy regimens for breast cancer patients on the left side, and the findings can be used to select appropriate radiotherapy programs for breast cancer patients.

Single-cell transcriptomics reveals cellular heterogeneity and macrophage-to-mesenchymal transition in bicuspid calcific aortic valve disease.

BACKGROUND: Bicuspid aortic valve (BAV) is the most prevalent congenital valvular heart defect, and around 50% of severe isolated calcific aortic valve disease (CAVD) cases are associated with BAV. Although previous studies have demonstrated the cellular heterogeneity of aortic valves, the cellular composition of specific BAV at the single-cell level remains unclear. METHODS: Four BAV specimens from aortic valve stenosis patients were collected to conduct single-cell RNA sequencing (scRNA-seq). In vitro experiments were performed to further validate some phenotypes. RESULTS: The heterogeneity of stromal cells and immune cells were revealed based on comprehensive analysis. We identified twelve subclusters of VICs, four subclusters of ECs, six subclusters of lymphocytes, six subclusters of monocytic cells and one cluster of mast cells. Based on the detailed cell atlas, we constructed a cellular interaction network. Several novel cell types were identified, and we provided evidence for established mechanisms on valvular calcification. Furthermore, when exploring the monocytic lineage, a special population, macrophage derived stromal cells (MDSC), was revealed to be originated from MRC1+ (CD206) macrophages (Macrophage-to-Mesenchymal transition, MMT). FOXC1 and PI3K-AKT pathway were identified as potential regulators of MMT through scRNA analysis and in vitro experiments. CONCLUSIONS: With an unbiased scRNA-seq approach, we identified a full spectrum of cell populations and a cellular interaction network in stenotic BAVs, which may provide insights for further research on CAVD. Notably, the exploration on mechanism of MMT might provide potential therapeutic targets for bicuspid CAVD.

Prognostic and recurrent significance of SII in patients with pancreatic head cancer undergoing pancreaticoduodenectomy.

Background: To investigate the clinical significance of preoperative inflammatory status in patients with pancreatic head carcinoma (PHC), we performed a single-center study to assess it. Method: We studied a total of 164 patients with PHC undergoing PD surgery (with or without allogeneic venous replacement) from January 2018 to April 2022. Systemic immune-inflammation index (SII) was the most important peripheral immune index in predicting the prognosis according to XGBoost analysis. The optimal cutoff value of SII for OS was calculated according to Youden index based on the receiver operating characteristic (ROC) curve and the cohort was divided into Low SII group and High SII group. Demographic, clinical data, laboratory data, follow-up data variables were obtained and compared between the two groups. Kaplan-Meier curves, univariable and multivariable Cox regression models were used to determine the association between preoperative inflammation index, nutritional index and TNM staging system with OS and DFS respectively. Results: The median follow-up time was 16 months (IQR 23), and 41.4% of recurrences occurred within 1 year. The cutoff value of SII was 563, with a sensitivity of 70.3%, and a specificity of 60.7%. Peripheral immune status was different between the two groups. Patients in High SII group had higher PAR, NLR than those in Low SII group (P <0.01, <0.01, respectively), and lower PNI (P <0.01). Kaplan-Meier analysis showed significantly poorer OS and DFS (P < 0.001, <0.001, respectively) in patients with high SII. By using the multivariable Cox regression model, high SII (HR, 2.056; 95% CI, 1.082-3.905, P=0.028) was significant predictor of OS. Of these 68 high-risk patients who recurrence within one year, patients with widespread metastasis had lower SII and worse prognosis (P <0.01). Conclusion: High SII was significantly associated with poor prognosis in patients with PHC. However, in patients who recurrence within one year, SII was lower in patients at TNM stage III. Thus, care needs to be taken to differentiate those high-risk patients.

Green biosynthesis of rare DHA-phospholipids by lipase-catalyzed transesterification with edible algal oil in solvent-free system and catalytic mechanism study.

Docosahexaenoic acid (DHA)-enriched phosphatidylcholine (PC) has received significant scientific attention due to the health benefits in food and pharmaceutical products. In this work, the edible algal oil rich in DHA-triacylglycerol (DHA-TAG) without pretreatment was first used as the DHA donor for the transesterification of phospholipids (PLs) to prepare three kinds of rare PLs, including DHA-PC, DHA-phosphatidylethanolamine (DHA-PE), and DHA-phosphatidylserine (DHA-PS). Here, 153 protein structures of triacylglycerol lipase (EC 3.1.1.3) were virtually screened and evaluated by transesterification. PLA1 was the best candidate due to a higher DHA incorporation. Results showed that the transesterification of PC with DHA-TAG at 45°C and 0.7% water content (without additional water addition) could produce DHA-PC with 39.1% DHA incorporation at 30 min. The different DHA donors, including forms of fatty acid, methyl ester, and triglycerides, were compared. Molecular dynamics (MD) was used to illustrate the catalytic mechanism at the molecular level containing the diffusions of substrates, the structure-activity relationship of PLA1, and the effect of water content.

Self-actuated biomimetic nanocomposites for photothermal therapy and PD-L1 immunosuppression.

Biomimetic nanocomposites are widely used in the biomedical field because they can effectively solve the problems existing in the current cancer treatment by realizing multi-mode collaborative treatment. In this study, we designed and synthesized a multifunctional therapeutic platform (PB/PM/HRP/Apt) with unique working mechanism and good tumor treatment effect. Prussian blue nanoparticles (PBs) with good photothermal conversion efficiency were used as nuclei and coated with platelet membrane (PM). The ability of platelets (PLTs) to specifically target cancer cells and inflammatory sites can effectively enhance PB accumulation at tumor sites. The surface of the synthesized nanocomposites was modified with horseradish peroxidase (HRP) to enhance the deep penetration of the nanocomposites in cancer cells. In addition, PD-L1 aptamer and 4T1 cell aptamer AS1411 were modified on the nanocomposite to achieve immunotherapy and enhance targeting. The particle size, UV absorption spectrum and Zeta potential of the biomimetic nanocomposite were determined by transmission electron microscope (TEM), Ultraviolet-visible (UV-Vis) spectrophotometer and nano-particle size meter, and the successful preparation was proved. In addition, the biomimetic nanocomposites were proved to have good photothermal properties by infrared thermography. The cytotoxicity test showed that it had a good killing ability of cancer cells. Finally, thermal imaging, tumor volume detection, immune factor detection and Haematoxilin-Eosin (HE) staining of mice showed that the biomimetic nanocomposites had good anti-tumor effect and could trigger immune response in vivo. Therefore, this biomimetic nanoplatform as a promising therapeutic strategy provides new inspiration for the current diagnosis and treatment of cancer.

A nomogram model to predict the portal vein thrombosis risk after surgery in patients with pancreatic cancer.

Background: Portal vein thrombosis (PVT) is a common postoperative complication in patients with pancreatic cancer (PC), significantly affecting their quality of life and long-term prognosis. Our aim is to establish a new nomogram to predict the risk of PVT after PC surgery. Method: We collected data from 416 patients who underwent PC surgery at our hospital between January 2011 and June 2022. This includes 87 patients with PVT and 329 patients without PVT. The patients were randomly divided into a training group and a validation group at a ratio of 7:3. We constructed a nomogram model using the outcomes from both univariate and multivariate logistic regression analyses conducted on the training group. The nomogram's predictive capacity was assessed using calibration curve, receiver operating characteristic (ROC) curve, and decision curve analysis (DCA). Results: In the study, the prevalence of PVT was 20.9%. Age, albumin, vein reconstruction and preoperative D-dimer were independent related factors. The model achieved a C-index of 0.810 (95% confidence interval: 0.752-0.867), demonstrating excellent discrimination and calibration performance. The area under the ROC curve of the nomogram was 0.829 (95% CI: 0.750-0.909) in the validation group. DCA confirmed that the nomogram model was clinically useful when the incidence of PVT in patients was 5%-60%. Conclusion: We have established a high-performance nomogram for predicting the risk of PVT in patients undergoing PC surgery. This will assist clinical doctors in identifying individuals at high risk of PVT and taking appropriate preventive measures.

Effects of probiotic supplementation on related side effects after chemoradiotherapy in cancer patients.

Objectives: Chemotherapy and radiotherapy generally cause serious adverse side effects in cancer patients, thereby affecting subsequent treatment. Numerous studies have shown that taking probiotics is an option for preventing and treating these side effects. In this investigation, a meta-analysis of the effects of oral probiotics on side effects brought on by radiotherapy, chemotherapy, or chemoradiotherapy treatment will be carried out. Methods: Two researchers independently and carefully reviewed all pertinent studies that were published before June 30, 2022 and were accessible on PubMed, Embase, Cochrane Library, and the Web of Science. Moreover, the Cochrane Collaboration's Tool was used to evaluate the risk of bias. Utilizing Review Manager software version 5.4, data were retrieved from eligible studies to evaluate their merits and determine odds ratios (OR) and 95% confidence intervals (CIs) (RevMan 5.4). Results: 2 097 patients from 16 randomized controlled trials were extracted, and standard meta-analysis methods were used to examine the data. Compared with the placebo groups, oral probiotics significantly reduced the side effects caused by radiotherapy and chemotherapy on various types of cancer, such as head and neck cancer, pelvic and abdominal cancer, breast cancer, lung cancer, etc. (OR: 0.31, 95% CI: 0.20 - 0.48; P < 0.005). Further analysis found that the incidence of diarrhea in patients with pelvic and abdominal cancers (OR: 0.32, 95% CI: 0.16 - 0.65; P < 0.005) and the frequency of oral mucositis in patients with head and neck tumors were also significantly lower (OR: 0.28, 95% CI: 0.18 - 0.43; P < 0.005) after the oral administration of probiotics. This suggests that probiotics have a positive influence on the treatment of side effects after chemoradiotherapy. Additionally, a funnel plot revealed that there was no significant publication bias in this study. Conclusions: Probiotics may help to reduce the occurrence of cancer therapy-related side effects, especially oral mucositis in head and neck tumors and diarrhea in patients with pelvic and abdominal tumors. However, given the small number of clinical trials involved, additional randomized, double-blind, multicentric trials in a larger population are required. This paper may assist researchers in improving trial design in the selection of probiotic strains and selecting appropriate patients who may benefit from probiotic treatments.

Nano tantalum-coated 3D printed porous polylactic acid/beta-tricalcium phosphate scaffolds with enhanced biological properties for guided bone regeneration.

Bone defects caused by tumors section, traffic accidents, and surgery remain a challenge in clinical. The drawbacks of traditional autografts and allografts limit their clinical application. 3D printed porous scaffolds have monumental potential to repair bone defects but still cannot effectively promote bone formation. Nano tantalum (Ta) has been reported with effective osteogenesis capability. Herein, we fabricated 3D printed PLA/ß-TCP scaffold by using the fused deposition modeling (FDM) technique. Ta was doped on the surface of scaffolds utilizing the surface adhesion ability of polydopamine to improve its properties. The constructed PLA/ß-TCP/PDA/Ta had good physical properties. In vitro studies demonstrated that the PLA/ß-TCP/PDA/Ta scaffolds considerably promote cell proliferation and migration, and it additionally has osteogenic properties. Therefore, Ta doped 3D printed PLA/ß-TCP/PDA/Ta scaffold could incontestably improve surface bioactivity and lead to better osteogenesis, which may provide a unique strategy to develop bioactive bespoke implants in orthopedic applications.

The efficient and green synthesis of biodiesel from crude oil without degumming catalyzed by sodium carbonate supported MoS2.

The transesterification of lecithin with methanol catalyzed by 23 kinds of alkaline salts was investigated for the preparation of biodiesel. Sodium carbonate was confirmed as the best catalyst due to its excellent catalytic performance, environmental friendliness, and great stability. Next, it was successfully immobilized on the surface of hierarchical nanosheets of MoS2. The prepared catalyst was characterized via XRD, FTIR, SEM, and TEM techniques. After immobilization, the highest specific activity reached 40.58 ± 0.78 U mgNa2CO3 -1, which was 2.43 times higher than that of unsupported Na2CO3. Meanwhile, the highest yield reached 99.8%. The excellent performance of the supported catalysts was attributed to a synergistic effect between MoS2 and the absorbed sodium carbonate. Firstly, sodium carbonate was uniformly dispersed on the surface of MoS2 to minimize the mass transfer resistance. Secondly, the electron-rich outer layer of MoS2 promoted the deprotonation of methanol to form methoxy anions. The prepared catalyst was further applied in the transesterification of lecithin-containing triglycerides to prepare fatty acid methyl esters (FAMEs). The experimental results showed that the addition of lecithin would promote the transesterification of triglycerides. The yields of FAMEs were close to 100% in all cases when the lecithin content was increased from 1% to 40%. Hence, this supported sodium carbonate catalyst should be a promising candidate for biodiesel production from crude oil without degumming.

Developing a novel calcium magnesium silicate/graphene oxide incorporated silk fibroin porous scaffold with enhanced osteogenesis, angiogenesis and inhibited osteoclastogenesis.

Recently, biofunctional ions (Mg2+, Si4+, etc) and graphene derivatives are proved to be promising in stimulating bone formation. In this study, a novel inorganic/organic composite porous scaffold based on silk fibroin (SF), graphene oxide (GO), and calcium magnesium silicate (CMS) was developed for bone repair. The porous scaffolds obtained by lyophilization showed a little difference in pore structure while GO and CMS displayed a good interaction with SF matrix. The addition of CMS with good mineralization potential and sustainedly release ability of biofunctional ions (Ca2+, Mg2+and Si4+) increased the strength of SF scaffolds a little and facilitated the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) by upregulating bone formation-related genes (ALP, COL1, OC and Runx2). The further incorporation of GO in SF scaffolds enhanced the compressive strength and water retention, and also remarkably promoted the osteogenic differentiation of BMSCs. Besides, the angiogenesis of human umbilical vein endothelial cells was significantly promoted by CMS/GO/SF scaffold extract through the upregulation of angiogenesis genes (eNOs and bFGF). Moreover, the osteoclastic formation ability of RAW264.7 cells was suppressed by the released ions from CMS/GO/SF scaffold through the down-regulation of CAK, MMP9 and TRAP. The promoted osteogenesis, angiogenesis and inhibited osteoclastogenesis functions of CMS/GO/SF composite scaffold may enable it as a novel therapy for bone repair and regeneration.

Anti-Photoaging and Anti-Inflammatory Effects of Ginsenoside Rk3 During Exposure to UV Irradiation.

Ginseng is a widely cultivated perennial plant in China and Korea. Ginsenoside Rk3 is one of the major active components of ginseng and is a promising candidate to regulate skin pigments and exert anti-photoaging effects on skin physiology. Ginsenoside Rk3 was mixed with a cream (G-Rk3 cream) and smeared on the skin of mice. Then, the mice were exposed to ultraviolet (UV) A (340 nm and 40 W) and UVB (313 nm and 40 W) radiation. Special attention was given to the anti-photoaging and anti-inflammatory effects of ginsenoside Rk3 on the mouse skin. Macroscopic evaluation indicated that the mouse dorsal skin looked smooth and plump even under UV irradiation for 12 weeks. Pathological analysis indicated that there was no obvious photoaging or inflammation in the mouse skin that was treated with the G-Rk3 cream. More healthy, intact, and neat collagen fibers were observed in mice treated with the G-Rk3 cream than in untreated mice. Further analysis proved that ginsenoside Rk3 could inhibit the decrease in water and hydroxyproline levels in skin tissues and the loss of superoxide dismutase and glutathione peroxidase activities in the blood. Moreover, ginsenoside Rk3 slowed or halted increases in malondialdehyde, matrix metalloproteinase (MMP)-1, and MMP-3 levels in the blood and levels of interleukin 1, interleukin 6, and tumor necrosis factor α in skin tissues. In conclusion, ginsenoside Rk3 plays a significant role in inhibiting photoaging and inflammation to protect skin health.

Identification of intact peptides by top-down peptidomics reveals cleavage spots in thermolabile wine proteins.

Prevention of haze formation in wines is challenging for winemakers. Thermolabile proteins in wines, notably thaumatin-like proteins (TLPs) and chitinases (CHIs), undergo structural changes under varying physicochemical conditions, resulting in protein aggregation and visible haze in bottled products. Peptidases are an alternative fining method, although an effective proteolysis under typical winemaking conditions (acidic pH and low temperature) is difficult to achieve. In this study, tryptic peptides from TLPs and CHIs were identified by MS-based peptidomics (top-down proteomics) after exposure of scissile bonds on the protein surface. As proposed by the theory of limited proteolysis, protein conformational changes following temperature and pH variations allowed the detection of enzyme-accessible regions. Protein structure visualization and molecular dynamics simulations were used to highlight cleavage spots and provide the scientific basis for haze formation mechanisms. The described method offers a tool to the search for ideal enzymes to prevent wine haze.

ALICE: a hybrid AI paradigm with enhanced connectivity and cybersecurity for a serendipitous encounter with circulating hybrid cells.

A fully automated and accurate assay of rare cell phenotypes in densely-packed fluorescently-labeled liquid biopsy images remains elusive. Methods: Employing a hybrid artificial intelligence (AI) paradigm that combines traditional rule-based morphological manipulations with modern statistical machine learning, we deployed a next generation software, ALICE (Automated Liquid Biopsy Cell Enumerator) to identify and enumerate minute amounts of tumor cell phenotypes bestrewed in massive populations of leukocytes. As a code designed for futurity, ALICE is armed with internet of things (IOT) connectivity to promote pedagogy and continuing education and also, an advanced cybersecurity system to safeguard against digital attacks from malicious data tampering. Results: By combining robust principal component analysis, random forest classifier and cubic support vector machine, ALICE was able to detect synthetic, anomalous and tampered input images with an average recall and precision of 0.840 and 0.752, respectively. In terms of phenotyping enumeration, ALICE was able to enumerate various circulating tumor cell (CTC) phenotypes with a reliability ranging from 0.725 (substantial agreement) to 0.961 (almost perfect) as compared to human analysts. Further, two subpopulations of circulating hybrid cells (CHCs) were serendipitously discovered and labeled as CHC-1 (DAPI+/CD45+/E-cadherin+/vimentin-) and CHC-2 (DAPI+ /CD45+/E-cadherin+/vimentin+) in the peripheral blood of pancreatic cancer patients. CHC-1 was found to correlate with nodal staging and was able to classify lymph node metastasis with a sensitivity of 0.615 (95% CI: 0.374-0.898) and specificity of 1.000 (95% CI: 1.000-1.000). Conclusion: This study presented a machine-learning-augmented rule-based hybrid AI algorithm with enhanced cybersecurity and connectivity for the automatic and flexibly-adapting enumeration of cellular liquid biopsies. ALICE has the potential to be used in a clinical setting for an accurate and reliable enumeration of CTC phenotypes.

Mild microwave ablation combined with HSP90 and TGF­ß1 inhibitors enhances the therapeutic effect on osteosarcoma.

Osteosarcoma is the most common malignant bone tumour and the second leading cause of cancer­related death in children and adolescents. Microwave ablation has an excellent therapeutic effect on bone tumours by instantaneously increasing the temperature in the tumour; however, there is a risk of damaging the surrounding healthy tissues by exposure to a high temperature when the treatment power is too large. In the present study, two anti­tumour reagents, a heat shock protein 90 (HSP90) inhibitor (PF­04929113) and a transforming growth factor­ß1 (TGF­ß1) inhibitor (SB­525334) were employed to enhance the therapeutic effect of mild­power microwave ablation. It was revealed that microwaving to 48˚C combined with HSP90 and TGF­ß1 inhibitors significantly increased the apoptotic rate of VX2 cells. The same results were observed during in vivo experiments using New Zealand rabbits to model osteosarcoma. In addition, the results indicated that the expression of cytochrome c, caspase­3 and caspase­9 were upregulated in response to the treatment, which indicated that the mitochondrial apoptotic signalling pathway had been activated. These findings may provide a novel strategy for the development of microwave ablation in osteosarcoma treatment, which could effectively kill tumour cells without damaging the surrounding normal tissues.

Biofunctions of antimicrobial peptide-conjugated alginate/hyaluronic acid/collagen wound dressings promote wound healing of a mixed-bacteria-infected wound.

The increase in severe infections caused by antibiotic drug resistance and the decrease in the number of new antibacterial drugs approved for use in the last few decades are driving the need for the development of new antimicrobial strategies. Antimicrobial peptides (AMPs) are a potential new class of antimicrobial drugs that are expected to solve the problem of global antibiotic drug resistance. Herein, the AMP Tet213 was immobilised onto the substrates of alginate (ALG), hyaluronic acid (HA), and collagen (COL) to form the ALG/HA/COL-AMP wound dressing. This wound dressing exhibited a high degree of swelling and the appropriate porosity, mechanical properties, and biodegradability. The Tet213-immobilised ALG/HA/COL dressings exhibited antimicrobial activity against three pathogenic bacterial strains (Gram-negative E. coli and Gram-positive MRSA and S. aureus) and facilitated the proliferation of NIH 3T3 fibroblast cells. In addition, the ALG/HA/COL-AMP antimicrobial dressings promoted wound healing, re-epithelialisation, collagen deposition, and angiogenesis. Moreover, the wound-healing effects of ALG/HA/COL-AMP surpassed the gauze and ALG/HA/COL compared to commercially available silver-based dressings (Aguacel Ag). These results suggest that the Tet213-conjugated ALG/HA/COL wound dressing, with its multiple biological activities, is a promising wound-dressing material.

Molecular detection of fungal communities in the Hawaiian marine sponges Suberites zeteki and Mycale armata.

Symbiotic microbes play a variety of fundamental roles in the health and habitat ranges of their hosts. While prokaryotes in marine sponges have been broadly characterized, the diversity of sponge-inhabiting fungi has barely been explored using molecular approaches. Fungi are an important component of many marine and terrestrial ecosystems, and they may be an ecologically significant group in sponge-microbe interactions. This study tested the feasibility of using existing fungal primers for molecular analysis of sponge-associated fungal communities. None of the eight selected primer pairs yielded satisfactory results in fungal rRNA gene or internal transcribed spacer (ITS) clone library constructions. However, 3 of 10 denaturing gradient gel electrophoresis (DGGE) primer sets, which were designed to preferentially amplify fungal rRNA gene or ITS regions from terrestrial environmental samples, were successfully amplified from fungal targets in marine sponges. DGGE analysis indicated that fungal communities differ among different sponge species (Suberites zeteki and Mycale armata) and also vary between sponges and seawater. Sequence analysis of DGGE bands identified 23 and 21 fungal species from each of the two sponge species S. zeteki and M. armata, respectively. These species were representatives of 11 taxonomic orders and belonged to the phyla of Ascomycota (seven orders) and Basidiomycota (four orders). Five of these taxonomic orders (Malasseziales, Corticiales, Polyporales, Agaricales, and Dothideomycetes et Chaetothyriomcetes incertae sedis) have now been identified for the first time in marine sponges. Seven and six fungal species from S. zeteki and M. armata, respectively, are potentially new species because of their low sequence identity (< or =98%) with their references in GenBank. Phylogenetic analysis indicated sponge-derived sequences were clustered into "marine fungus clades" with those from other marine habitats. This is the first report of molecular analysis of fungal communities in marine sponges, adding depth and dimension to our understanding of sponge-associated microbial communities.

Karyotype analysis of Gossypium arboreum x G. bickii by genome in situ hybridization.

By using genome in situ hybridization (GISH) on root somatic chromosomes of allotetraploid derived from the cross Gossypium arboreum x G. bickii with genomic DNA (gDNA) of G. bickii as a probe, two sets of chromosomes, consisting of 26 chromosomes each, were easily distinguished from each other by their distinctive hybridization signals. GISH analysis directly proved that the hybrid G. arboreum x G. bickii is an allotetraploid amphiploid. The karyotype formula of the species was 2n = 4x = 52 = 46m (4sat) + 6sm (4sat). We identified four pairs of satellites with two pairs in each sub-genome. FISH analysis using 45S rDNA as a probe showed that the cross G. arboreum x G. bickii contained 14 NORs. At least five pairs of chromosomes in the G sub-genome showed double hybridization (red and blue) in their long arms, which indicates that chromatin introgression from the A sub-genome had occurred.