Nanofiber orientation and surface functionalization modulate human mesenchymal stem cell behavior in vitro

Electrospun nanofiber meshes have emerged as a new generation of scaffold membranes possessing a number of features suitable for tissue regeneration. One of these features is the flexibility to modify their structure and composition to orchestrate specific cellular responses. In this study, we investigated the effects of nanofiber orientation and surface functionalization on human mesenchymal stem cell (hMSC) migration and osteogenic differentiation. We used an in vitro model to examine hMSC migration into a cell-free zone on nanofiber meshes and mitomycin C treatment to assess the contribution of proliferation to the observed migration. Poly (ɛ-caprolactone) meshes with oriented topography were created by electrospinning aligned nanofibers on a rotating mandrel, while randomly oriented controls were collected on a stationary collector. Both aligned and random meshes were coated with a triple-helical, type I collagen-mimetic peptide, containing the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif. Our results indicate that nanofiber GFOGER peptide functionalization and orientation modulate cellular behavior, individually, and in combination. GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes. Aligned nanofiber meshes displayed increased cell migration along the direction of fiber orientation compared to random meshes; however, fiber alignment did not influence osteogenic differentiation. Compared to each other, GFOGER coating resulted in a higher proliferation-driven cell migration, whereas fiber orientation appeared to generate a larger direct migratory effect. This study demonstrates that peptide surface modification and topographical cues associated with fiber alignment can be used to direct cellular behavior on nanofiber mesh scaffolds, which may be exploited for tissue regeneration.

Evidence for the role of Mycobacteriumtuberculosis RecG helicase in DNA repair and recombination

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacteriumtuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M.tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichiacoli recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions.

Trans-dichlorooxovandium (IV) complex as a novel photoinducible DNA interstrand crosslinker for cancer therapy

Although DNA interstrand crosslinking (ICL) agents such as mitomycin C, cisplatin and psoralen serve as potent anticancer drugs, these agents are known to have dose-limiting toxic effects on normal cells. Moreover, tumor resistance to these agents has been reported. Here, we show that trans-dichlorooxovanadium (IV) complex of pyrenyl terpyridine (VDC) is a novel photoinducible DNA crosslinking agent. By a combination of in vitro and ex vivo experiments including plasmid-based assays, we find that VDC forms monoadducts on the DNA and can be activated by UV-A and visible light to generate DNA interstrand crosslinks. VDC efficiently activates Fanconi anemia (FA) pathway of DNA interstrand crosslink repair. Strikingly, photoinduction of VDC induces prolonged activation of cell cycle checkpoint and a high degree of cell death in homologous recombination (HR)/ICL repair defective cells. Moreover, VDC specifically targets cells that express pathological RAD51C mutants. These data imply that VDC can be potentially used for cancer therapy and suggest that tumors arising in patients with gene mutations in FA and HR repair pathway can be specifically targeted by a photoactivatable VDC.

I. Studies on bacteriophage DNA's and minicircular DNA's in M. lysodeikticus and E. coli 15. II. Flow dichroism of DNA solutions

Part I

Chapter 1.....A physicochemical study of the DNA molecules from the three bacteriophages, N1, N5, and N6, which infect the bacterium, M. lysodeikticus, has been made. The molecular weights, as measured by both electron microscopy and sedimentation velocity, are 23 x 10⁶ for N5 DNA and 31 x 10⁶ for N1 and N6 DNA's. All three DNA's are capable of thermally reversible cyclization. N1 and N6 DNA's have identical or very similar base sequences as judged by membrane filter hybridization and by electron microscope heteroduplex studies. They have identical or similar cohesive ends. These results are in accord with the close biological relation between N1 and N6 phages. N5 DNA is not closely related to N1 or N6 DNA. The denaturation T_m of all three DNA's is the same and corresponds to a (GC) content of 70%. However, the buoyant densities in CsCl of Nl and N6 DNA's are lower than expected, corresponding to predicted GC contents of 64 and 67%. The buoyant densities in Cs₂SO₄ are also somewhat anomalous. The buoyant density anomalies are probably due to the presence of odd bases. However, direct base composition analysis of N1 DNA by anion exchange chromatography confirms a GC content of 70%, and, in the elution system used, no peaks due to odd bases are present.

Chapter 2.....A covalently closed circular DNA form has been observed as an intracellular form during both productive and abortive infection processes in M. lysodeikticus. This species has been isolated by the method of CsC1-ethidium bromide centrifugation and examined with an electron microscope.

Chapter 3.....A minute circular DNA has been discovered as a homogeneous population in M. lysodeikticus. Its length and molecular weight as determined by electron microscopy are 0.445 μ and 0.88 x 10⁶ daltons respectively. There is about one minicircle per bacterium.

Chapter 4.....Several strains of E. coli 15 harbor a prophage. Viral growth can be induced by exposing the host to mitomycin C or to uv irradiation. The coliphage 15 particles from E. coli 15 and E, coli 15 T^- appear as normal phage with head and tail structure; the particles from E. coli 15 TAU are tailless. The complete particles exert a colicinogenic activity on E.coli 15 and 15 T^-, the tailless particles do not. No host for a productive viral infection has been found and the phage may be defective. The properties of the DNA of the virus have been studied, mainly by electron microscopy. After induction but before lysis, a closed circular DNA with a contour length of about 11.9 μ is found in the bacterium; the mature phage DNA is a linear duplex and 7.5% longer than the intracellular circular form. This suggests the hypothesis that the mature phage DNA is terminally repetitious and circularly permuted. The hypothesis was confirmed by observing that denaturation and renaturation of the mature phage DNA produce circular duplexes with two single-stranded branches corresponding to the terminal repetition. The contour length of the mature phage DNA was measured relative to φX RFII DNA and λ DNA; the calculated molecular weight is 27 x 10⁶. The length of the single-stranded terminal repetition was compared to the length of φX 174 DNA under conditions where single-stranded DNA is seen in an extended form in electron micrographs. The length of the terminal repetition is found to be 7.4% of the length of the nonrepetitious part of the coliphage 15 DNA. The number of base pairs in the terminal repetition is variable in different molecules, with a fractional standard deviation of 0.18 of the average number in the terminal repetition. A new phenomenon termed "branch migration" has been discovered in renatured circular molecules; it results in forked branches, with two emerging single strands, at the position of the terminal repetition. The distribution of branch separations between the two terminal repetitions in the population of renatured circular molecules was studied. The observed distribution suggests that there is an excluded volume effect in the renaturation of a population of circularly permuted molecules such that strands with close beginning points preferentially renature with each other. This selective renaturation and the phenomenon of branch migration both affect the distribution of branch separations; the observed distribution does not contradict the hypothesis of a random distribution of beginning points around the chromosome.

Chapter 5....Some physicochemical studies on the minicircular DNA species in E. coli 15 (0.670 μ, 1.47 x 10⁶ daltons) have been made. Electron microscopic observations showed multimeric forms of the minicircle which amount to 5% of total DNA species and also showed presumably replicating forms of the minicircle. A renaturation kinetic study showed that the minicircle is a unique DNA species in its size and base sequence. A study on the minicircle replication has been made under condition in which host DNA synthesis is synchronized. Despite experimental uncertainties involved, it seems that the minicircle replication is random and the number of the minicircles increases continuously throughout a generation of the host, regardless of host DNA synchronization.

Part II

The flow dichroism of dilute DNA solutions (A₂₆₀≈0.1) has been studied in a Couette-type apparatus with the outer cylinder rotating and with the light path parallel to the cylinder axis. Shear gradients in the range of 5-160 sec.^-1 were studied. The DNA samples were whole, "half," and "quarter" molecules of T4 bacteriophage DNA, and linear and circular λb₂b₅c DNA. For the linear molecules, the fractional flow dichroism is a linear function of molecular weight. The dichroism for linear A DNA is about 1.8 that of the circular molecule. For a given DNA, the dichroism is an approximately linear function of shear gradient, but with a slight upward curvature at low values of G, and some trend toward saturation at larger values of G. The fractional dichroism increases as the supporting electrolyte concentration decreases.

草鱼体内肾细胞姐妹染色单体分化及交换的初步研究

本文确立了一个以草鱼体内肾细胞姐妹染色单体交换频率为指标的检测环境诱变或致癌物质的短期试验系统。采用硫堇-UV-Giemsa染色法,分析了草鱼体内肾细胞的SCD-2(注射BrdU后第二个细胞周期的中期分裂相的SCD)频率和SCE频率。用500微克/克体重BrdU体内标记5天,草鱼肾细胞SCD-2频率为8.58±0.22%;SCE频率为3.05±2.523 SCE_5/细胞。以丝裂霉素C(Mitomycin C,MMC)作为阳性对照,分析了化合物亚硝基胍(N-methyl-N~1-nitro-N-nitro

Induction and preliminary characterization of a novel halophage SNJ1 from lysogenic Natrinema sp F5

Halophage SNJ1 was induced with mitomycin C from Natrinema sp. strain F5. The phage produces plaques on Natrinema sp. strain J7 only. The phage has a head of about 67 nm in diameter and a tail of 570 nm in length and belongs morphologically to the family Siphoviridae. The phage is strongly salt dependent; NaCl concentration affects the integrity of SNJ1, phage adsorption, and plaque formation. The optimal NaCl concentration for phage adsorption and plaque formation is 30% and 25%, respectively.

Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing

In this study, an in vitro multicellular tumor spheroid model was developed using microencapsulation, and the feasibility of using the microencapsulated. multicellular tumor spheroid (MMTS) to test the effect of chemotherapeutic drugs was investigated. Human MCF-7 breast cancer cells were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and a single multicellular spheroid 150 mu m in diameter was formed in the microcapsule after 5 days of cultivation. The cell morphology, proliferation, and viability of the MMTS were characterized using phase contrast microscopy, BrdU-Iabeling, MTT stain, calcein AM/ED-2 stain, and H&E stain. It demonstrated that the MMTS was viable and that the proliferating cells were mainly localized to the periphery of the cell spheroid and the apoptotic cells were in the core. The MCF-7 MMTS was treated with mitomycin C (MC) at a concentration of 0.1, 1, or 10 times that of peak plasma concentration (ppc) for up to 72 h. The cytotoxicity was demonstrated. clearly by the reduction in cell spheroid size and the decrease in cell viability. The MMTS was further used to screen the anticancer effect of chemotherapeutic drugs, treated with MC, adriamycin (ADM) and 5-fluorouracil (5-FU) at concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h. MCF-7 monolayer culture was used as control. Similar to monolayer culture, the cell viability of MMTS was reduced after treatment with anticancer drugs. However, the inhibition rate of cell viability in MMTS was much lower than that in monolayer culture. The MMTS was more resistant to anticancer drugs than monolayer culture. The inhibition rates of cell viability were 68.1%, 45.1%, and 46.8% in MMTS and 95.1%, 86.8%, and 91.6% in monolayer culture treated with MC, ADM, and 5-FU at 10 ppc for 72 h, respectively. MC showed the strongest cytotoxicity in both MMTS and monolayer, followed by 5-FU and ADM. It demonstrated that the MMTS has the potential to be a rapid and valid in vitro model to screen chemotherapeutic drugs with a feature to mimic in vivo three-dimensional (3-D) cell growth pattern.

Atypical Fibroxanthoma of the Bulbar Conjunctiva.

PURPOSE: To report a rare case of atypical fibroxanthoma (AFX) of the bulbar conjunctiva, and to compare it with previously published cases of conjunctival AFX. METHODS: A 37-year-old woman developed a growth on the bulbar conjunctiva of her left eye that increased in size and redness over 4 months and was associated with blurry vision in the left eye, occasional diplopia, irritation of the eye, and increasing tearing. The mass was surgically excised. RESULTS: Slit-lamp examination disclosed a highly vascularized conjunctival lesion with intact lustrous epithelium and a raised nodular edge encroaching on the nasal corneal limbus of the left eye. Pathological examination and immunohistochemistry were diagnostic of AFX. CONCLUSIONS: AFX of the conjunctiva is rare, with this being only the fifth example of this neoplasm reported at this site. Complete surgical excision is the most appropriate treatment option.

Adjuvant portal-vein infusion of fluorouracil and heparin in colorectal cancer: A randomised trial

Background. There is conflicting evidence on the efficacy of regional adjuvant chemotherapy, via portal-vein infusion (PVI), after resection of colorectal cancer. We undertook a randomised controlled multicentre trial to investigate the efficacy of PVI (500 mg/m2 fluorouracil plus 5000 IU heparin daily for 7 days). Methods. 1235 of about 1500 potentially eligible patients were randomly assigned surgery plus PVI or surgery alone (control). The patients were followed up for a median of 63 months, with yearly screening for recurrent disease. The primary endpoint was survival; analyses were by intention to treat. Findings. 619 patients in the control group and 616 in the PVI group met eligibility criteria. 164 (26%) control-group patients and 173 (28%) PVI-group patients died. 5-year survival did not differ significantly between the groups (73 vs 72%; 95% CI for difference -6 to 4). The control and PVI groups were also similar in terms of disease-free survival at 5 years (67 vs 65%) and the number of patients with liver metastases (79 vs 77%). Interpretation. PVI of fluorouracil, at a dose of 500 mg/m2 for 7 days, cannot be recommended as the sole adjuvant treatment for high-risk colorectal cancer after complete surgical excision. However, these results cannot eliminate a small benefit when PVI is used at a higher dosage or in combination with mitomycin.

Identification of a methylation hotspot in the death receptor Fas/CD95 in bladder cancer

We characterized Fas immunoreactivity, functionality and its role in the response to mitomycin-C (MMC) chemotherapy in vitro in cell lines and in vivo in bladder washings from 23 transitional cell carcinoma of the bladder (TCCB) patients, harvested prior to and during MMC intravesical treatment. Having established the importance of functional Fas, we investigated the methylation and exon 9 mutation as mechanisms of Fas silencing in TCCB. For the first time, we report p53 up-regulation in 9/14 and Fas up-regulation in 7/9 TCCB patients during intravesical MMC treatment. Fas immunoreactivity was strong in the TCCB cell line T24 and in 17/20 (85%) tumor samples from patients with advanced TCCB. T24 and HT1376 cells were resistant to MMC and recombinant Fas ligand, whilst RT4 cells were responsive to Fas ligand and MMC. Using RT4 cells as a model, siRNA targeting p53 significantly reduced MMC-induced p53 and Fas up-regulation and stable DN-FADD transfection decreased MMC-induced apoptosis, suggesting that functional Fas enhances chemotherapy responses in a p53-dependent manner. In HT1376 cells, 5-aza-2-deoxycytidine (12 µM) induced Fas immunoreactivity and reversed methylation at CpG site -548 within the Fas promoter. This site was methylated in 13/24 (54%) TCCB patient samples assessed using Methylation-Specific Polymerase Chain Reaction. There was no methylation at either the p53 enhancer region within the first intron or at the SP-1 binding region in the promoter and no mutation within exon 9 in tumor DNA extracted from 38 patients. Methylation at CpG site -548 is a potential target for demethylating drugs.

Plasmid pRTL1 controlling 1-chloroalkane degradation by Rhodococcus rhodochrous NCIMB 13064

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and use a wide range of 1-haloalkanes as sole carbon and energy source. The 1-chloroalkane degradation phenotype may be lost by cells spontaneously or after treatment with Mitomycin C. Two laboratory derivatives of the original strain exhibited differing degrees of stability of the chloroalkane degradation marker. Plasmids of approximately 100 kbp (pRTL1) and 80 kbp (pRTL2) have been found in R. rhodochrous NCIMB13064. pRTL1 was shown to be carrying at least some genes for the dehalogenation of 1-chloroalkanes with short chain lengths (C-3 to C-9). However, no connection was found between the utilization of 1-chloroalkanes with longer chain lengths (C-12 to C-18) and the presence of pRTL1. Three separate events were observed to lead to the inability of NCIMB13064 to dehalogenate the short-chain 1-chloroalkanes; the complete loss of pRTL1, the integration of pRTL1 into the chromosome, or the deletion of a 20-kbp fragment in pRTL1. High-frequency transfer of the 1-chloroalkane degradation marker associated with pRTL1 has been demonstrated in bacterial crosses between different derivatives of R. rhodochrous NCIMB13064, (C) 1995 Academic Press, Inc.

Effect of technique on intraocular pressure after combined cataract and glaucoma surgery: An evidence-based review.

TOPIC:

To analyze the literature pertaining to the techniques used in combined cataract and glaucoma surgery, including the technique of cataract extraction, the timing of the surgery (staged procedure versus combined procedure), the anatomic location of the operation, and the use of antifibrosis agents.

CLINICAL RELEVANCE:

Cataract and glaucoma are both common conditions and are often present in the same patient. There is no agreement concerning the optimal surgical management of these disorders when they coexist.

METHODS/LITERATURE REVIEWED:

Electronic searches of English language articles published since 1964 were conducted in Pub MED and CENTRAL, the Cochrane Collaboration's database. These were augmented by a hand search of six ophthalmology journals and the reference lists of a sample of studies included in the literature review. Evidence grades (A, strong; B, moderate; C, weak; I, insufficient) were assigned to the evidence that involved a direct comparison of alternative techniques.

RESULTS:

The preponderance of evidence from the literature suggests a small (2-4 mmHg) benefit from the use of mitomycin-C (MMC), but not 5-fluorouracil (5-FU), in combined cataract and glaucoma surgery (evidence grade B). Two-site surgery provides slightly lower (1-3 mmHg) intraocular pressure (IOP) than one-site surgery (evidence grade C), and IOP is lowered more (1-3 mmHg) by phacoemulsification than by nuclear expression in combined procedures (evidence grade C). There is insufficient evidence to conclude either that staged or combined procedures give better results or that alternative glaucoma procedures are superior to trabeculectomy in combined procedures.

CONCLUSIONS:

In the literature on surgical techniques and adjuvants used in the management of coexisting cataract and glaucoma, the strongest evidence of efficacy exists for using MMC, separating the incisions for cataract and glaucoma surgery, and removing the nucleus by phacoemulsification.

Corneal hysteresis is correlated with reduction in axial length after trabeculectomy.

BACKGROUND: We sought to determine whether corneal biomechanical parameters are predictive of reduction in axial length after anti-metabolite trabeculectomy. METHODS: Chinese subjects undergoing trabeculectomy with mitomycin C by a single experienced surgeon underwent the following measurements: Corneal hysteresis (CH, Ocular Response Analyzer, Reichert Ophthalmic Instruments), Goldmann intra-ocular pressure (IOP), central corneal thickness (CCT) and axial length (AL, IOLMaster, Carl Zeiss Meditec, Dublin, CA) were measured pre-operatively, and AL, CH and IOP were measured 1 day and 1 week post-operatively. RESULTS: Mean age of 31 subjects was 52.0 ± 15.2 years, and 15 (48.4%) were female. The mean pre-operative IOP of 21.4 ± 9.3 mmHg was reduced to 8.2 ± 4.6 mmHg 1 day and 11.0 ± 4.4 mmHg 1 week post-operatively (p < 0.001). AL declined from 22.99 ± 0.90 to 22.76 ± 0.87 mm at 1 day and 22.74 ± 0.9 mm at 1 week; 30/31 (%) eyes had a decline in AL (p < 0.001, sign test). In multivariate linear regression models including post-operative data from 1 day and 1 week, greater decline in Goldmann IOP (p < 0.0001, greater pre-op axial length (p < 0.001) and lower pre-operative CH (p = 0.03), were all associated with greater decline in post-operative axial length. CONCLUSIONS: Eyes with lesser ability of the ocular coat to absorb energy (lower CH) had significantly greater decrease in axial length after trabeculectomy-induced IOP-lowering.

Erwinia amylovora bacteriophage resistance

It has been proposed that phages can be used commercially as a biopesticide for the control of fire blight caused by the phytopathogen Erwinia amylovora. The aim of these studies was to investigate two common bacterial resistance mechanisms, lysogeny and exopolysaccharide production and their influence on phage pathogenesis. A multiplex real-time PCR protocol was designed to monitor and quantify Podoviridae and Myoviridae phages. This protocol is compatible with known E. amylovora and Pantoea agglomerans rtPCR primers/probes which allowed simultaneous study of both phage and bacterial targets. Using in vitro positive phage selection, bacteriophage insensitive derivatives were isolated within sensitive populations of E. amylovora. Prophage screening with real-time PCR and mitomycin C induction determined that the insensitive derivatives harboured the temperate Podoviridae phage ΦEaTlOO. Lysogenic conversion resulted in resistance to secondary homologous phage infections. Prophage screening of environmental samples of E. amylovora and P. agglomerans collected from various locations in Canada, United States and Europe did not demonstrate lysogeny. Therefore, lysogeny is rare or absent while these bacterial species reside on the plant. Recombineering was used to construct exopolysaccharide deficient E. amylovora mutants. The EPS amylovoran mutants became resistant to Podoviridae and certain Siphoviridae phages. Increasing amylovoran production increased phage population growth, presumably by increasing the total number of bacterial cell surface receptors which promoted increased phage infections. In contrast, amylovoran did not playa role in Myoviridae infections, nor did production of the EPS levan for any phage pathogenesis.

Approches thérapeutiques de la carcinose péritonéale d’origine appendiculaire récidivante

Objectif: Le traitement de la carcinose péritonéale (CP) d’origine appendiculaire récidivante demeure sous-étudié. L’objectif est d’évaluer le traitement à offrir aux patients présentant une récidive de la maladie. Méthodologie: Depuis 2003, les données de tous les patients avec CP d’origine appendiculaire traités dans notre centre ont été recueillies. Le traitement de la maladie primaire consistait en une cytoréduction chirurgicale complète de la CP suivie d’une chimiothérapie hyperthermique intra-péritoneale (CHIP) à l’oxaliplatine. Lors d’une récidive, une deuxième cytoréduction avec une CHIP à la mitomycine C était offerte aux patients rencontrant certains critères de sélection. Résultats: Un total de 58 patients ont subi une cytoréduction complète suivie d’une CHIP avec l’oxaliplatine. Durant la période de suivi, 22 patients démontrèrent une maladie récidivante (39%). Le péritoine était le site de récidive le plus fréquent. Les survies sans maladie étaient statistiquement différentes selon le grade histopathologique et le degré de complétude de la cytoréduction. Parmi ces 22 patients, huit patients ont reçu une deuxième cytoréduction suivie d’une CHIP à la mitomycine C, un patient a subi une cytoréduction complète seule et 13 patients avaient une maladie non résécable. La survie globale moyenne des patients ayant reçu un deuxième traitement fut de 28 mois. Les taux de morbidité et de mortalité suite à une deuxième cytoréduction suivie d’une CHIP furent de 25% et 0%, respectivement. Conclusion: Une cytoréduction suivie d’une CHIP demeure une intervention possible chez les patients qui présentent une maladie récidivante, mais certains critères doivent être appliqués afin de maximiser la survie et minimiser la morbidité et la mortalité de cette approche.