Linear and Nonlinear Optics of Broad-Band Laser Pulses: Diffraction.

The typical spectrally limited laser pulse in the near-infrared region is narrow-band up to 40-50 fs. Its spectral width Δk is much smaller than the carrying wavenumber k0 (Δk ≪ k0) . For such kinds of pulses, on distances of a few diffraction lengths, the diffraction is of a Fresnel's type and their evolution can be described correctly in the frame of the well-known paraxial evolution equation. The technology established in 1985 of amplification through chirping of laser pulses triggered remarkable progress in laser optics along with the construction of femtosecond (fs) laser facilities producing high intensity fields of the order of 1015-1021 W/cm2. However, the duration of the pulse was quickly shortened from picoseconds down to 5-6 fs, which have a broad-band nature (Δk ∼ k0). The linear and nonlinear propagation dynamics of broad-band pulses is quite different form their narrow-band counterparts. Here, we review the appropriate theoretical approach to study the evolution of the pulse. Moreover, we shed light on the different diffraction regimes inherent to both narrow-band and broad-band laser pulses and compare them to unveil the main differences. Using this very method, in subsequent papers, we will investigate the influence of the dispersion and nonlinearity on the laser pulse propagation in isotropic media.

BRCA1 and 53BP1 regulate reprogramming efficiency by mediating DNA repair pathway choice at replication-associated double-strand breaks.

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1.

Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size.

Limitations in cell proliferation are important for normal function of differentiated tissues and essential for the safety of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest or by impairing S phase entry or S phase completion and determined growth potential, differentiation, and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells toward insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.

Detection of base analogs incorporated during DNA replication by nanopore sequencing.

DNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no single method can identify the location, direction and speed of replication forks with high resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labeling of replicating or repaired DNA. Here, we demonstrate the use of the Oxford Nanopore Technologies MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU as well as EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show that the large adduct Biotin can be distinguished from the smaller analog IdU, which opens the possibility of using analog combinations to identify the location and direction of DNA synthesis. Furthermore, we detect IdU label on single DNA molecules in the genome of mouse pluripotent stem cells and using CRISPR/Cas9-mediated enrichment, determine replication rates using newly synthesized DNA strands in human mitochondrial DNA. We conclude that this novel method, termed Replipore sequencing, has the potential for on target examination of DNA replication in a wide range of biological contexts.

NanoMod: a computational tool to detect DNA modifications using Nanopore long-read sequencing data.

BACKGROUND: Recent advances in single-molecule sequencing techniques, such as Nanopore sequencing, improved read length, increased sequencing throughput, and enabled direct detection of DNA modifications through the analysis of raw signals. These DNA modifications include naturally occurring modifications such as DNA methylations, as well as modifications that are introduced by DNA damage or through synthetic modifications to one of the four standard nucleotides. METHODS: To improve the performance of detecting DNA modifications, especially synthetically introduced modifications, we developed a novel computational tool called NanoMod. NanoMod takes raw signal data on a pair of DNA samples with and without modified bases, extracts signal intensities, performs base error correction based on a reference sequence, and then identifies bases with modifications by comparing the distribution of raw signals between two samples, while taking into account of the effects of neighboring bases on modified bases ("neighborhood effects"). RESULTS: We evaluated NanoMod on simulation data sets, based on different types of modifications and different magnitudes of neighborhood effects, and found that NanoMod outperformed other methods in identifying known modified bases. Additionally, we demonstrated superior performance of NanoMod on an E. coli data set with 5mC (5-methylcytosine) modifications. CONCLUSIONS: In summary, NanoMod is a flexible tool to detect DNA modifications with single-base resolution from raw signals in Nanopore sequencing, and will facilitate large-scale functional genomics experiments that use modified nucleotides.

Avalanche parametric conversion and white spectrum generation from infrared femtosecond pulses in glasses.

The observation of discrete lines in the white spectrum at the initial stage of filamentation of powerful femtosecond laser pulses, propagating in silica glasses, as well as the filamentation without plasma channels observed in the experiments in air, pushed us to look for other nonlinear mechanisms for describing these effects. In this paper, we present a new parametric conversion mechanism for asymmetric spectrum broadening of femtosecond laser pulses towards higher frequencies in isotropic media. This mechanism includes cascade generation with THz spectral shift for solids and GHz shift for gases. The process works simultaneously with the four-photon parametric wave mixing. The theoretical model proposed agrees well with the experimental data.

Homologous recombination DNA repair genes play a critical role in reprogramming to a pluripotent state.

Induced pluripotent stem cells (iPSCs) hold great promise for personalized regenerative medicine. However, recent studies show that iPSC lines carry genetic abnormalities, suggesting that reprogramming may be mutagenic. Here, we show that the ectopic expression of reprogramming factors increases the level of phosphorylated histone H2AX, one of the earliest cellular responses to DNA double-strand breaks (DSBs). Additional mechanistic studies uncover a direct role of the homologous recombination (HR) pathway, a pathway essential for error-free repair of DNA DSBs, in reprogramming. This role is independent of the use of integrative or nonintegrative methods in introducing reprogramming factors, despite the latter being considered a safer approach that circumvents genetic modifications. Finally, deletion of the tumor suppressor p53 rescues the reprogramming phenotype in HR-deficient cells primarily through the restoration of reprogramming-dependent defects in cell proliferation and apoptosis. These mechanistic insights have important implications for the design of safer approaches to creating iPSCs.

Comparative analysis of diagnostic methods in meniscal lesions.

UNLABELLED: The aim of this study is to determine the accuracy of clinical and MRI diagnosis in comparison with arthroscopy for detection of meniscal lesions. Also, to answer if MRI diagnosis impacts on the decision of the surgeon for the choice of treatment (operative or conservative). MATERIAL AND METHODS: We examined 70 patients with knee injuries. Clinical diagnosis was established using the case-history of the patient and positive clinical tests for meniscal injuries (McMurray and Aplay). All patients underwent MRI on a 1.5 T magnet for MRI diagnosis. This was followed by arthroscopy for final diagnosis. Clinical and MRI diagnoses were correlated with the arthroscopic diagnosis which was used as a gold standard. RESULTS: Of 70 patients with knee injuries, 55 had a clinical diagnosis of meniscal lesions out of whom 44 patients had a medial meniscal lesion and 11 had a lateral meniscal lesion. Arthroscopy confirmed the clinical diagnosis in 32 patients (72.72%) (44 vs 32) in medial meniscal lesion, and 8 patients (72.7%) (11 vs 8) with a lateral meniscal lesion. In MRI diagnosis of 56 patients with medial meniscal lesion arthroscopy confirmed the diagnosis in 34 patients (60.7%) (56 vs 34) and pf 10 patients with lateral meniscal lesion arthroscopy confirmed the diagnosis in 6 patients (60%) (10 vs 6). The sensitivity, specificity, PPV and NPV of clinical diagnosis versus MRI for medial meniscus were (79.9% vs 79.5%); (58.1% vs 38.1%); (69.8% vs 69.6%); (69.2% vs 69.2%). The sensitivity, specificity, PPV and NPV of clinical diagnosis versus MRI for lateral meniscus were (50% vs 40%); (92.7% vs 92.7%); (63.6% vs 60%); (87.9% vs 85.5%). CONCLUSIONS: Carefully performed clinical examination can give an equal or better diagnosis of meniscal lesions in comparison with MRI diagnosis. Any experienced orthopaedic surgeon can trust his clinical diagnosis as an indication of arthroscopy. When the clinical diagnosis is established, with no doubts due to positivity of the clinical tests, the MRI is not essential. In suspected cases where there is a dilemma, MRI is very helpful in making a decision for arthroscopy. The diagnostic accuracy of clinical and MRI diagnosis of meniscal lesions is high. Their reliability in diagnosing meniscal lesions is evident. lesion, clinical diagnosis, MRI, arthroscopy.

Electromagnetic shock wave in nonlinear vacuum: exact solution.

An analytical approach to the theory of electromagnetic waves in nonlinear vacuum is developed. The evolution of the pulse is governed by a system of nonlinear wave vector equations. An exact solution with its own angular momentum in the form of a shock wave is obtained.

Three cases of paralytic poliomyelitis associated with type 3 vaccine poliovirus strains in Bulgaria.

Oral poliovirus vaccine (OPV) can cause, in extremely rare cases vaccine-associated paralytic poliomyelitis in recipients, or contacts of vaccinees. Three cases of vaccine-associated paralytic poliomyelitis (two contacts and one recipient) occurred in the Bourgas region of Bulgaria in the spring of 2006. The first two cases, notified as acute flaccid paralysis, were 55 days old unvaccinated twin brothers, having been in contact with vaccinees. The third case concerned a 4-month-old infant who had received the first OPV dose 37 days prior to the onset of illness. Complete clinical, epidemiological, virological, serological and molecular investigations of the children with paralysis and their contacts were undertaken. In all the three cases type 3 polioviruses were isolated from fecal samples and characterized as Sabin-like poliovirus strains. Type 3 polioviruses isolated from the twin brothers demonstrated by sequence analysis U-to-C back mutation at nt 472 of the 5' UTR, known to correlate with neurovirulence, and mutation in the VP1 region. Type 3 poliovirus isolated from the third child demonstrated in the 3D sequenced region a recombination with Sabin type 1 poliovirus. In the latter region, three silent mutations and one, resulting in amino acid substitution, were also observed. The clinical, epidemiological and virological data and the neurological sequelae observed 60 days following the onset of paralysis, confirmed the diagnosis of vaccine-associated paralytic poliomyelitis in all the three patients.

On the possible role of surface elasticity in emulsion stability.

We have measured the short-time and long-time elastic responses to compression of various types of surfactant layers adsorbed at oil-water interfaces. We prepared reasonably monodisperse oil-in-water emulsions with the same surfactants and monitored the time evolution of the emulsion droplets' diameter. We used a broad variety of surfactants (cationic, nonionic, and small polymers) and alkanes with different chain lengths. The emulsion drop size evolution is first controlled by Ostwald ripening and later on by drop coalescence, the later step being quite short. The overall emulsion lifetime is therefore dominated by ripening and for a given oil appears well correlated with the low-frequency surface elasticity as expected (and not with the high-frequency one, which is expected to control coalescence). When the oil chain length is changed, the stability is related more to the oil solubility in water, which also controls ripening. The overall results demonstrate the great importance of surface elasticity in emulsion stability.