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PubMed2004

Indications for peripheral light-chain revision and somatic hypermutation without a functional B-cell receptor in precursors of a composite diffuse large B-cell and Hodgkin's lymphoma.

Composite lymphomas are rare combinations of Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma in the same patient, where clonal relatedness has been observed in most of the few cases analyzed. Here, we report a composite classical HL and diffuse large B-cell lymphoma (DLBCL) with interesting molecular features. Micromanipulation of single cells and analysis of V gene rearrangements revealed clonal relatedness with shared and distinct mutations, indicative of derivation from a common germinal center (GC) B-cell precursor and also of further development of both lymphomas in a GC. In the DLBCL, a very high mutation load, including inactivating mutations, and two copies of the same clonal rearrangement with different mutations in single cells were observed. Intriguingly, in the DLBCL precursor somatic hypermutation activity continued after acquisition of destructive V gene mutations, a feature previously found only in Epstein-Barr virus (EBV) infected B-cell expansions. Furthermore, we found evidence of light-chain receptor revision in the lymphoma precursor during a GC reaction. Re-expression of the V(D)J recombination machinery may enhance genomic instability in GC B cells and contribute to lymphomagenesis.

Aged↗
PubMed2003

Structural transitions and elasticity from torque measurements on DNA.

Knowledge of the elastic properties of DNA is required to understand the structural dynamics of cellular processes such as replication and transcription. Measurements of force and extension on single molecules of DNA have allowed direct determination of the molecule's mechanical properties, provided rigorous tests of theories of polymer elasticity, revealed unforeseen structural transitions induced by mechanical stresses, and established an experimental and conceptual framework for mechanical assays of enzymes that act on DNA. However, a complete description of DNA mechanics must also consider the effects of torque, a quantity that has hitherto not been directly measured in micromanipulation experiments. We have measured torque as a function of twist for stretched DNA--torsional strain in over- or underwound molecules was used to power the rotation of submicrometre beads serving as calibrated loads. Here we report tests of the linearity of DNA's twist elasticity, direct measurements of the torsional modulus (finding a value approximately 40% higher than generally accepted), characterization of torque-induced structural transitions, and the establishment of a framework for future assays of torque and twist generation by DNA-dependent enzymes. We also show that cooperative structural transitions in DNA can be exploited to construct constant-torque wind-up motors and force-torque converters.

DNA↗
PubMed2004

A three-dimensional optical photonic crystal with designed point defects.

Photonic crystals offer unprecedented opportunities for miniaturization and integration of optical devices. They also exhibit a variety of new physical phenomena, including suppression or enhancement of spontaneous emission, low-threshold lasing, and quantum information processing. Various techniques for the fabrication of three-dimensional (3D) photonic crystals--such as silicon micromachining, wafer fusion bonding, holographic lithography, self-assembly, angled-etching, micromanipulation, glancing-angle deposition and auto-cloning--have been proposed and demonstrated with different levels of success. However, a critical step towards the fabrication of functional 3D devices, that is, the incorporation of microcavities or waveguides in a controllable way, has not been achieved at optical wavelengths. Here we present the fabrication of 3D photonic crystals that are particularly suited for optical device integration using a lithographic layer-by-layer approach. Point-defect microcavities are introduced during the fabrication process and optical measurements show they have resonant signatures around telecommunications wavelengths (1.3-1.5 microm). Measurements of reflectance and transmittance at near-infrared are in good agreement with numerical simulations.

Journal Article↗
PubMed2006

Human embryonic stem cell lines derived from single blastomeres.

The derivation of human embryonic stem (hES) cells currently requires the destruction of ex utero embryos. A previous study in mice indicates that it might be possible to generate embryonic stem (ES) cells using a single-cell biopsy similar to that used in preimplantation genetic diagnosis (PGD), which does not interfere with the embryo's developmental potential. By growing the single blastomere overnight, the resulting cells could be used for both genetic testing and stem cell derivation without affecting the clinical outcome of the procedure. Here we report a series of ten separate experiments demonstrating that hES cells can be derived from single blastomeres. In this proof-of-principle study, multiple biopsies were taken from each embryo using micromanipulation techniques and none of the biopsied embryos were allowed to develop in culture. Nineteen ES-cell-like outgrowths and two stable hES cell lines were obtained. The latter hES cell lines maintained undifferentiated proliferation for more than eight months, and showed normal karyotype and expression of markers of pluripotency, including Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, nanog and alkaline phosphatase. These cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. The ability to create new stem cell lines and therapies without destroying embryos would address the ethical concerns of many, and allow the generation of matched tissue for children and siblings born from transferred PGD embryos.

Blastomeres↗
PubMed2002

Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA.

Analysis of transcript representation on gene microarrays requires microgram amounts of total RNA or DNA. Without amplification, such amounts are obtainable only from millions of cells. However, it may be desirable to determine transcript representation in few or even single cells in aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells. Nucleic-acid amplification methods could be used in these cases, but it is difficult to amplify different transcripts in a sample without distorting quantitative relationships between them. Linear isothermal RNA amplification has been used to amplify as little as 10 ng of total cellular RNA, corresponding to the amount obtainable from thousands of cells, while still preserving the original abundance relationships. However, the available procedures require multiple steps, are labor intensive and time consuming, and have not been shown to preserve abundance information from smaller starting amounts. Exponential amplification, on the other hand, is a relatively simple technology, but is generally considered to bias abundance relationships unacceptably. These constraints have placed beyond current reach the secure and routine application of microarray analysis to single or small numbers of cells. Here we describe results obtained with a rapid and highly optimized global reverse transcription#150;PCR (RT-PCR) procedure. Contrary to prevalent expectations, the exponential approach preserves abundance relationships through amplification as high as 3 x 10(11)-fold. Further, it reduces by a million-fold the input amount of RNA needed for microarray analysis, and yields reproducible results from the picogram range of total RNA obtainable from single cells.

Cluster Analysis↗
PubMed2003

Microassembly of semiconductor three-dimensional photonic crystals.

Electronic devices and their highly integrated components formed from semiconductor crystals contain complex three-dimensional (3D) arrangements of elements and wiring. Photonic crystals, being analogous to semiconductor crystals, are expected to require a 3D structure to form successful optoelectronic devices. Here, we report a novel fabrication technology for a semiconductor 3D photonic crystal by uniting integrated circuit processing technology with micromanipulation. Four- to twenty-layered (five periods) crystals, including one with a controlled defect, for infrared wavelengths of 3-4.5 microm, were integrated at predetermined positions on a chip (structural error <50 nm). Numerical calculations revealed that a transmission peak observed at the upper frequency edge of the bandgap originated from the excitation of a resonant guided mode in the defective layers. Despite their importance, detailed discussions on the defective modes of 3D photonic crystals for such short wavelengths have not been reported before. This technology offers great potential for the production of optical wavelength photonic crystal devices.

Crystallization↗
PubMed1996

Radiofrequency as a lesioning model in experimental spinal cord injury.

Many models have been developed to study spinal cord injury (SCI), such as cryogenic lesioning, hot water injury, scalpel lesioning, compressive trauma using clips, electromechanical devices, extradural cuffs, and weight-drop techniques. In this study, the radiofrequency (RF) lesion was used for inducing an experimental SCI in cats. The neuropathology was correlated with the MRI. In this model, 4 cats were injured at the thoracic spinal cord (T11-T12) with a lesion of 65 degrees C for 1 min using a micromanipulated penetrating RF electrode. The MRI of the lesions after 2, 3, 5, and 6 weeks post-injury as well as the correlative histological changes were obtained. The RF-induced lesion was discrete with little spreading across the spinal cord. There was a good correlation between the histopathology findings and the MRI. We conclude that experimental RF lesioning of the spinal cord can produce a consistent lesion with predictable histopathological changes in experimental animals. A 65 degree C injury for 1 min induced a clinical picture of an incomplete SCI. The RF lesioning should be considered as a new model to study SCI, particularly those with a penetrating component.

Animals↗
PubMed2000

Stromal cell-independent differentiation of human cord blood CD34+CD38- lymphohematopoietic progenitors toward B cell lineage.

To study the cytokine regulation of early stages of human B-lymphopoiesis, we developed a stroma-free two-step culture system. Single human cord blood CD34+CD38- cells were individually cultured by micromanipulation with interleukin (IL)-3, stem cell factor (SCF), fIt3 ligand (FL), IL-6 and granulocyte colony-stimulating factor (G-CSF). About 10% of the cells formed primary colonies, which were individually tested for myeloid and B-lymphoid potentials by reculturing aliquots of the primary colony cells into secondary myeloid and B-lymphoid cultures. One third of the primary colonies proved capable of differentiation into CD19+IgM+ cells, as well as into myeloid lineage cells. RT-PCR analyses revealed that some cells in the primary culture had already matured to express B cell-specific transcripts. Thus, the combination of IL-3, SCF, FL, IL-6 and G-CSF supported the differentiation of CD34+CD38- lymphohematopoietic progenitors toward B cell lineage in addition to myeloid lineages. Screening of cytokines to identify the minimum requirement of cytokines in the primary culture revealed that IL-3 and SCF were essential and that the addition of FL, and to a lesser extent IL-6 or G-CSF, to the combination of IL3 and SCF remarkably enhanced the primary colony formation and the generation of CD19+ cells in the secondary B-lymphoid culture.

Antigens, CD34↗
PubMed1998

Ejaculatory stimulation, quality of semen and reproductive aspects in spinal cord injured men.

this is a retrospective study of 39 male spinal cord injured patients who, over the last 5 years, have consulted us for ejaculatory dysfunction or infertility. All of these patients underwent at least two distinct sessions during which attempts were made to induce an ejaculation by vibratory stimulation, electric stimulation, or by using subcutaneous physostigmine. A semen analysis was performed each time that an ejaculate was obtained. By one or more of these techniques, 75% of these subjects were able to ejaculate. The level of the neurological lesion had a significant influence on the success rate. Likewise, the mode employed to produce an ejaculation influenced certain characteristics of the sperm collected (mean volume, percentage of motile forms), the best results being obtained by vibratory stimulation. Among the 10 couples who had consulted us for medically assisted reproduction, three pregnancies were obtained, two of which resulted in the birth of healthy children. Although the techniques of stimulation that we use allow ejaculation, the possibilities of reproduction remain limited in particular because of the difficulty in obtaining semen of adequate quality. Nevertheless, recent methods of micromanipulation of gametes should considerably improve the prognosis of the seminal insufficiencies in men with spinal cord injury.

Adult↗
PubMed1999

A comprehensive approach to the management of male infertility following spinal cord injury.

OBJECTIVES: To review the outcomes of management of male infertility following spinal cord injury in a specialised fertility clinic. STUDY DESIGN: Retrospective review of medical records. SETTING: The Fertility Clinic of a 45-bed spinal cord injury service in New South Wales, Australia. METHODS: The medical and fertility clinic records of 113 males who attended the clinic between 1987 and 1997 were reviewed. RESULTS: The rates of semen retrieval using vibroejaculation and electroejaculation were 67% and 97% respectively. Thirty-one individuals (37%) and their partners sought assistance with the primary aim of achieving a pregnancy. Intravaginal insemination (IVI) undertaken at home following vibroejaculation resulted in a pregnancy rate per cycle of 22%. Electroejaculation and IVI proved less successful with a pregnancy rate per cycle of only 5%, although with intrauterine insemination (IUI) this rate improved to 30%. Micromanipulation in vitro fertilisation (IVF) procedures, primarily intracytoplasmic sperm injection, were used in 18 couples resulting in a pregnancy rate per cycle of 19%. In the 31 couples there have been a total of 17 pregnancies in 97 cycle attempts for an overall pregnancy rate per cycle of 18% and a cumulative pregnancy rate per couple of 55%. Twelve of the pregnancies have resulted in 14 live births (including two sets of twins), there were three pregnancies ongoing at the date of review and there have been two spontaneous abortions. CONCLUSION: The benefits of a specialised fertility clinic offering a comprehensive, client-focused approach with education, fertility assessment and a range of semen retrieval and assisted reproduction options, are highlighted.

Adolescent↗
PubMed2001

Treatment of male infertility due to spinal cord injury using rectal probe electroejaculation: the Israeli experience.

STUDY DESIGN: Male infertility caused by anejaculation is common among patients with spinal cord injury (SCIP). The fertility options for SCIP have improved impressively over the past 10 years. We present the Israeli experience in the treatment of infertility in a large series of SCIP. The issues which are addressed include the treatment of ejaculatory dysfunction, seminal quality and fertility management in SCIP. SETTING: Sexual rehabilitation clinic, Neuro-Rehabilitation department, Sheba Medical Center, Israel. METHODS: Between June 1992 and May 1998, a total of 84 consecutive SCIP were treated in our clinic with electro-ejaculation (EEJ), representing a sample of the SCIP population, composed mostly of young men traumatically injured. The patients have sustained different levels and completeness of spinal injury. Among the patients 33 were interested in achieving pregnancy (39.3%), while the rest were interested in determining fertility potential for family. With EEJ, a low-current stimulation of the ejaculatory organs via a rectal probe is done. The collected semen is used for fertility determination or for fertilization. RESULTS: Eighty-four patients were treated by EEJ. Mean age was 31.3 and mean age at injury was 21.7. There were 29 cervical, 50 thoracic and five lumbar lesions. Sixty-three had complete injury (ASIA A) and 21 incomplete (ASIA B -15, ASIC C -5, ASIA D -1). Fifty-nine had upper motor neuron lesions, and 25 had lower motor neuron. A total of 355 stimulations were performed. Ejaculate was obtained in all patients in 350 stimulations (98.6%), and sperm was present in 74 patients (88.1%) in 296 of the stimulations (83.4%). Fairly good numbers of spermatozoa were obtained, whereas sperm motility and morphology of spermatozoa were low in most cases. A significant difference in sperm count, motility and morphology was noted between antegrade and retrograde samples. No significant improvement in sperm quality after four repeated consecutive stimulations was noted in 38 SCIP. Side effects were minor and encountered in 16 patients (19.1%). Out of 33 couples who wished to achieve pregnancy, 26 reached the stage of insemination. Four pregnancies were achieved after 33 cycles of In-Uterine-Insemination (pregnancy rate 28.6% per couple), and 15 after 68 cycles of In-Vitro-Fertilization (micromanipulation) (pregnancy rate of 68.75% per couple). In all, of 101 conception attempts 23 were successful, resulting in pregnancies in 18 couples, and accounting for an overall pregnancy rate of 70% per couple. CONCLUSION: The high percentage of pregnancies imply that, despite the typically poor sperm motility noted in EEJ, rectal probe EEJ combined with assisted reproductive techniques, and performed by a team approach, is an efficient and safe technique for treating infertility among SCIP.

Adult↗
PubMed2002

Electrowetting-based actuation of droplets for integrated microfluidics.

The serviceability of microfluidics-based instrumentation including 'lab-on-a-chip' systems critically depends on control of fluid motion. We are reporting here an alternative approach to microfluidics based upon the micromanipulation of discrete droplets of aqueous electrolyte by electrowetting. Using a simple open structure, consisting of two sets of opposing planar electrodes fabricated on glass substrates, positional and formational control of microdroplets ranging in size from several nanoliters to several microliters has been demonstrated at voltages between 15-100 V. Since there are no permanent channels or structures between the plates, the system is highly flexible and reconfigurable. Droplet transport is rapid and efficient with average velocities exceeding 10 cm s(-1) having been observed. The dependence of the velocity on voltage is roughly independent of the droplet size within certain limits, thus the smallest droplets studied (approximately 3 nl) could be transported over 1000 times their length per second. Formation, mixing, and splitting of microdroplets was also demonstrated using the same microactuator structures. Thus, electrowetting provides a means to achieve high levels of functional integration and flexibility for microfluidic systems.

Journal Article↗
PubMed2000

Changes in reactivity of rat arteries subjected to dynamic stretch.

The effect of dynamic stretch on the reactivity of the rat tail and mesenteric artery segments was studied. Segments mounted on a myograph were stretched by a computer-controlled motorized micromanipulator. Dynamic stretch (1, 5 or 7 Hz) inhibited the artery constriction induced by noradrenaline (10 microM), 5-hydroxytryptamine (0.7 microM), or electrical field stimulation of intramural nerves. In contrast, dynamic stretch enhanced the tetrodotoxin-insensitive dilation induced by electrical field stimulation of noradrenaline-contracted arteries. Maximal increase of dilation evoked by electrical field stimulation (24.5 +/- 5.0% in mesenteric and 50.3 +/- 15.6% in the tail artery) was observed at a dynamic stretch-frequency of 5 Hz. An inhibitor of nitric oxide synthesis, NG-nitro-L-arginine (100 microM), abolished the difference in reactivity between static and dynamic conditions. The results indicate that dynamic stretch of the arteries activates nitric oxide synthesis/secretion, thus reducing constrictor and increasing dilator responses to the stimuli used.

Acetylcholine↗
PubMed1999

X-chromosome inactivation analysis of isolated Reed-Sternberg cells in nodular sclerosing Hodgkin's disease.

In spite of several studies of immunoglobulin (Ig) gene rearrangements in whole Hodgkin's disease (HD) tissues and in isolated single Reed-Sternberg (RS) cells, the issue of clonality of the RS cell in HD remains incompletely resolved. Analysis of X-chromosome inactivation patterns (XCIPs) can be used to determine whether cell populations are clonal in origin. By PCR amplification of the human androgen receptor (HUMARA) loci using nested primers, we have studied XCIPs in six cases of HD of the nodular sclerosing (NS) subtype in which individual RS cells were isolated by micromanipulation from formalin-fixed, paraffin-embedded tissue sections immunostained with CD30 or CD15. In order to assess whether a clonal population of RS cells might be present in NSHD tissues, we compared the XCIPs obtained from whole NSHD tissues with those obtained from single RS cells harvested from the same tissues. Whole tissues from all six cases of NSHD showed balanced HUMARA allelic patterns, whereas an average of 83% (range 77-91%) of single RS cells from each of the six tissues expressed the same high- or low-molecular weight allele, suggesting that a clonal population of RS cells was likely to be present in each case. These data are consistent with the presence of a clonal population of RS cells in NSHD tissues.

Adult↗
PubMed1997

Analysis of V(H) genes rearranged by individual B cells in dermal infiltrates of patients with mycosis fungoides.

In patients with cutaneous T cell lymphomas such as mycosis fungoides B cells can frequently be detected in the lymphocytic dermal infiltrate. To analyse their immunoglobulin heavy chain gene repertoire, single B cells were obtained from tissue sections of two typical patients with mycosis fungoides using hydraulic micromanipulation followed by specific amplification of the respective gene segments by single-cell polymerase chain reaction (PCR) technique. A total of 21 V(H)DJ(H) genes was sequenced. From each individual B cell a single productive V(H)DJ(H) rearrangement was obtained. There was no clonal relationship detected between any of these rearrangements suggesting polyclonality of the infiltrating B cells. The representation of V(H) families was in accordance with the germ-line complexity. A remarkably high number of V(H) genes (5/13 in patient 1; 3/8 in patient 2) was completely or nearly germ-line-identical. Five of seven V(H)4 family genes were nearly unmutated. On the other hand, most of the V(H)3 gene family members were somatically mutated in an antigen-driven manner. The proportion of germ-line-identical V(H) genes, the usage of individual V(H), D, J(H) gene elements, and the pattern of somatic mutations found in the B cells infiltrating skin lesions of patients with mycosis fungoides resembles the peripheral blood repertoire, suggesting a bystander role of these cells.

Amino Acid Sequence↗
PubMed2000

The loose coupling mechanism in molecular machines of living cells.

Living cells have molecular machines for free energy conversion, for example, sliding machines in muscle and other cells, flagellar motors in bacteria, and various ion pumps in cell membranes. They are constructed from protein molecules and work in the nm (nanometer), pN (piconewton) and ms (millisecond) ranges, without inertia. In 1980s, a question was raised of whether the input-output or influx-efflux coupling in these molecular machines is tight or loose, and an idea of loose coupling was proposed. Recently, the long-distance multistep sliding of a single myosin head on an actin filament, coupled with the hydrolysis of one ATP molecule, was observed by Yanagida's group using highly developed techniques of optical microscopy and micromanipulation. This gave direct evidence for the loose coupling between the chemical reaction and the mechanical event in the sliding machine. In this review, I will briefly describe a historical overview of the input-output problem in the molecular machines of living cells.

Actins↗
PubMed2002

Application of a 1.48-microm diode laser for bisecting oocytes into two identical hemizonae for the hemizona assay.

Laser systems are very promising new technical tools in assisted reproduction. It was investigated if laser radiation can replace the mechanical cutting procedure via micromanipulator in the hemizona assay (HZA), a commonly used bioassay to determine the sperm-zona pellucida binding capacity. An oocyte was bisected precisely into two identical hemizonae with approximately 20 laser pulses (pulse length 30 msec) using a 1.48-microm diode laser. Compared with the conventional method using microscalpels for zona bisection, laser treated hemizonae showed equivalent sperm-binding and within the two groups there was no detectable difference between matching hemizonae in their capacity for tight sperm-binding. To evaluate whether laser radiation affects the outcome of the HZA when effects of certain substances are investigated, the spermatozoa were preincubated with human follicular fluid (hFF), which inhibits the binding of spermatozoa to zona pellucida in vitro. Supplementation with follicular fluid exerted an inhibitory effect in both groups. The hemizona index (HZI) showed no statistical differences between the two methods. Therefore, the 1.48-microm diode laser is a suitable new instrument for generating equally sized hemizonae. There is no use for holding pipettes and microscalpels, on the contrary, for performing the HZA the laser is a precise, very quick and easy to use new working tool.

Female↗
PubMed1997

Phenotypic and phylogenetic description of an Italian isolate of "Microthrix parvicella".

"Microthrix parvicella" strain RN1 was isolated from an activated sludge treatment plant in Italy using micromanipulation techniques. The strain grows as thin unbranchedfilaments which are Gram-positive with Neisser-positive granules. The isolate was characterized by analysis of the 16S rDNA which was amplified directly from cell biomass by the polymerase chain reaction and sequenced. "Microthrix parvicella" strain RN1 presents a very high similarity (100%) with another "M. parvicella" strain recently isolated in Australia, suggesting that this micro-organism, a novel, deep branching member of the actinomycetes subphylum, is the same causing the common events of bulking and foaming phenomena in activated sludge treatment plants throughout the world.

DNA, Bacterial↗