Mapping cellular responses to DNA double-strand breaks using CRISPR technologies.

DNA double-strand breaks (DSBs) are one of the most genotoxic DNA lesions, driving a range of pathological defects from cancers to immunodeficiencies. To combat genomic instability caused by DSBs, evolution has outfitted cells with an intricate protein network dedicated to the rapid and accurate repair of these lesions. Pioneering studies have identified and characterized many crucial repair factors in this network, while the advent of genome manipulation tools like clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) has reinvigorated interest in DSB repair mechanisms. This review surveys the latest methodological advances and biological insights gained by utilizing Cas9 as a precise 'damage inducer' for the study of DSB repair. We highlight rapidly inducible Cas9 systems that enable synchronized and efficient break induction. When combined with sequencing and genome-specific imaging approaches, inducible Cas9 systems greatly expand our capability to spatiotemporally characterize cellular responses to DSB at specific genomic coordinates, providing mechanistic insights that were previously unobtainable.

Achieving single nucleotide sensitivity in direct hybridization genome imaging.

Direct visualization of point mutations in situ can be informative for studying genetic diseases and nuclear biology. We describe a direct hybridization genome imaging method with single-nucleotide sensitivity, single guide genome oligopaint via local denaturation fluorescence in situ hybridization (sgGOLDFISH), which leverages the high cleavage specificity of eSpCas9(1.1) variant combined with a rationally designed guide RNA to load a superhelicase and reveal probe binding sites through local denaturation. The guide RNA carries an intentionally introduced mismatch so that while wild-type target DNA sequence can be efficiently cleaved, a mutant sequence with an additional mismatch (e.g., caused by a point mutation) cannot be cleaved. Because sgGOLDFISH relies on genomic DNA being cleaved by Cas9 to reveal probe binding sites, the probes will only label the wild-type sequence but not the mutant sequence. Therefore, sgGOLDFISH has the sensitivity to differentiate the wild-type and mutant sequences differing by only a single base pair. Using sgGOLDFISH, we identify base-editor-modified and unmodified progeroid fibroblasts from a heterogeneous population, validate the identification through progerin immunofluorescence, and demonstrate accurate sub-nuclear localization of point mutations.

Real-Time Measurement of Molecular Tension during Cell Adhesion and Migration Using Multiplexed Differential Analysis of Tension Gauge Tethers.

Cells must respond specifically and dynamically to mechanical cues from the extracellular environment and dysregulation of extracellular force sensing leads to a variety of diseases. Therefore, it is important to deconvolve the many inputs that transduce mechanical signals and understand how these signals are interpreted and responded to. DNA and peptide-based molecular force sensors have been previously developed to measure forces applied through single membrane receptors including integrins and Notch receptors. The tension gauge tether (TGT) exploits the physical rupture force of double-stranded DNA to measure and modulate the force applied through single receptor-ligand bonds and can cover a wide range of tension (10-60 pN). By exploiting a fluorescent dye-quencher pair and collecting differential fluorescence signals over time, we characterized the quenched tension gauge tether (qTGT) system and developed an image analysis protocol to measure molecular tension in quasi-real time. We show that this differential qTGT analysis method can simultaneously measure multiple levels of integrin-mediated molecular tension over a wide time scale during the onset of adhesion and cell migration.

Does anterolateral cage insertion enhance immediate stabilization of the functional spinal unit? A biomechanical investigation.

STUDY DESIGN: The three-dimensional flexibility of six human lumbar functional spinal units was measured after the anterolateral insertion of an interbody cage. OBJECTIVES: To determine whether an interbody cage inserted from an anterolateral direction stabilizes the spine with respect to the intact state and to compare the finding with that from the same cage inserted from an anterior direction. SUMMARY OF BACKGROUND DATA: Several biomechanical studies have shown that interbody cages do not stabilize the spine in extension. It is suspected that this may be caused by the destruction of the anterior longitudinal ligament and anterior anulus fibrosus. METHODS: Six human cadaveric lumbar functional spinal units were tested under pure moments of flexion, extension, bilateral axial rotation, and bilateral lateral bending to a maximum of 10 Nm. The relative intervertebral motions were measured by an optoelectronic camera system with the spinal units in the intact condition, after discectomy, after anterolateral interbody cage stabilization, and with additional translaminar screw fixation. The implant used was a central, porous, contoured implant with endplate fit. The results were compared with those of a previous study, which used the same implant inserted from an anterior direction. RESULTS: The anterolateral cage insertion significantly decreased the motion in comparison with the intact situation in flexion and lateral bending, but not in extension or axial rotation. No differences were found between the anterior and anterolateral insertion approaches in flexion or extension, but differences were observed in axial rotation and lateral bending, in which the anterolateral approach resulted in more motion. Additional translaminar screw fixation reduced motion to below intact levels in all loading directions. None of the surgical procedures introduced asymmetrical behavior. CONCLUSIONS: Anterolateral cage insertion did not stabilize the spine in extension or axial rotation and was not different from the anterior approach in flexion and extension. Additional translaminar screw fixation stabilized in all directions.

A transpedicularly implanted anterior spinal support.

This article describes a device that has been developed to augment posterior spinal fracture fixation by providing support for the anterior column of the spine. The device is contracted for insertion through the limited opening provided by the transpedicular approach to the vertebral body. Once in situ it is configured to support physiological loads. In this way the structural limitations of posterior instrumentation and the surgical difficulties of anterior spinal fixation instrumentation are overcome. A preliminary investigation of the new device together with in vitro studies of a prototype are described in this report. In vitro tests to determine the expansion characteristics and structural performance of the device were performed together with tests to determine the efficacy of the support to reduce loading on the posterior fixation device. It is shown that the device has the necessary expansion characteristics and can support physiological loads without failure. Tests using porcine vertebral columns with a simulated vertebral fracture showed a significant reduction in load carried by the posterior fixation plates (44 +/- 16%) when the anterior support was included in the construct. In addition, the difference in loading between the left and right plates was significantly reduced (81 +/- 12%).

Are indices of free radical damage related to exercise intensity.

The possibility that plasma levels of malonaldehyde (MDA) are altered by exercise has been examined. The presence of MDA has been recognized to reflect peroxidation of lipids resulting from reactions with free radicals. Maximal exercise, eliciting 100% of maximal oxygen consumption (VO2max) resulted in a 26% increase in plasma MDA (P less than 0.005). Short periods of intermittent exercise, the intensity of which was varied, indicated a correlation between lactate and MDA (r2 = 0.51) (p less than 0.001). Blood lactate concentrations increased throughout this exercise regimen. A significant decrease (10.3%) in plasma MDA occurred at 40% VO2max. At 70% VO2max plasma MDA was still below resting values, however the trend to an increase in MDA with exercise intensity was evident. At exhaustion, plasma MDA and lactate were significantly greater than at rest. These results suggest, that exhaustive maximal exercise induces free radical generation while short periods of submaximal exercise (i.e. less than 70% VO2max) may inhibit it and lipid peroxidation.

An improved glyoxylic acid technique for the histochemical localization of catecholamines in brown adipose tissue.

A glyoxylic acid method using cryostat sections to demonstrate catecholaminergic fibres of the central nervous system was modified to show the extent of the adrenergic innervation in rat brown adipose tissue. It revealed prominent interlacing fluorescent parenchymal fibres surrounding individual adipocytes. The density of this network of fine fibres was not evident using earlier techniques. The new method also confirmed the dense networks of adrenergic fibres associated with arterial vessels. Its specificity was verified by simultaneously performing radioenzymatic determinations of tissue catecholamine levels and histochemical studies of brown adipose tissue from normal and sympathectomized rats. Chemical sympathectomy with 6-hydroxydopamine resulted in a pronounced decrease in brown adipose tissue and heart catecholamine (noradrenalin and dopamine) levels. Significantly, in brown adipose tissue of sympathectomized animals no fluorescence could be detected in terminal nerves of either the parenchyma or those of vascular smooth muscles. Nevertheless, some intense fluorescence was seen in axon bundles. The findings suggest that catecholamines of the parenchymal innervation form a larger proportion of the total catecholamine content of brown adipose tissue than was previously believed, provide stronger support for direct control of the function of multilocular adipocytes, and also confirm unpublished data reporting considerable dopamine content in brown adipose tissue.

Abscisic Acid stimulation of suberization : induction of enzymes and deposition of polymeric components and associated waxes in tissue cultures of potato tuber.

Effect of abscisic acid (ABA) on suberization of potato (Solanum tuberosum var. Russet-Burbank) tuber tissue culture was studied by measuring deposition of suberin components and the level of certain key enzymes postulated to be involved in suberization. ABA treatment resulted in a 3-fold increase in the polymeric aliphatic components of suberin and a 4-fold increase in the polymeric aromatic components. Hydrocarbons and fatty alcohols, two components characteristic of waxes associated with potato suberin, increased 9- and 5-fold, respectively, as a result of ABA treatment. Thus, the deposition of the polymeric aliphatics and aromatics as well as waxes, all of which have been postulated to be components of suberized cell walls, was markedly stimulated by ABA. omega-Hydroxy-fatty acid dehydrogenase which showed a rather high initial level of activity increased only 60% due to ABA treatment. Phenylalanine ammonia-lyase activity reached a maximum at a 5-fold level after 4 days in the ABA medium, whereas the control showed only a 3-fold increase. ABA treatment also resulted in a dramatic (7-fold) increase in an isozyme of peroxidase which has been specifically associated with suberization. Thus, ABA appears to induce certain key enzymes which are most probably involved in suberization.

Biosynthesis, deposition, and partial characterization of potato suberin phenolics.

Alkaline nitrobenzene oxidation of the polymeric materials from wound-healed potato (Solanum tuberosum L. var. White Rose) tuber tissue liberated p-hydroxybenzaldehyde, vanillin, and minor amounts of syringaldehyde as determined by gas chromatography/mass spectrometry. The aromatic aldehydes were derived only from periderm. The amounts of aromatic aldehydes liberated were used as a measure of the deposition of phenolic suberin components. Phenolic deposition began after about 2 days of wound healing; after 8 days the amounts of p-hydroxybenzaldehyde released by nitrobenzene oxidation leveled off at 5 milligrams per gram dry weight and after 12 days vanillin liberation reached a maximum at 7.5 milligrams per gram dry weight. The time course of deposition of the phenolic polymeric material is analogous to that reported for the deposition of the aliphatic components of suberin and therefore these results are consistent with the proposed structure of suberin. Experiments with radiolabeled l-phenylalanine and cinnamic acid indicated that exogenous phenylalanine was less efficient than cinnamic acid as a precursor of suberin phenolics. Nitrobenzene oxidation of radiolabeled suberin preparations gave three major labeled fractions: a diethyl ether-soluble fraction containing aromatic aldehydes ( approximately 20%), an ethyl acetate-soluble fraction containing unknown compounds ( approximately 15%), and a condensed phenolic fraction ( approximately 10%). Thin-layer and gas-liquid chromatographic analysis of the ether fraction showed that the major labeled components were vanillin and p-hydroxybenzaldehyde. The condensed tannin fraction revealed the presence of several labeled macromolecular phenolic fractions. Elution profiles of the condensed tannin fraction from tissues suberized for different periods of time were essentially identical, suggesting qualitative similarity of deposition and polymerization of suberin phenolics throughout the duration of wound healing. Chlorogenic acid accumulation in wound healing potato tuber discs was measured by high-performance liquid chromatography. The level of this compound reached 130 micrograms per disk after 11 days and did not decline even after the deposition of suberin ceased, revealing no precursor role for this acid in suberization.

Hypoxia, smoking history, and exercise.

Six nonsmokers and six cigarette smokers, 22-34 years old, performed bicycle work (53% sea level VO2 max) for 30 min in an altitude chamber under four conditions: SL, simulated sea level (PIO2 = 159 torr, PB = 523 torr) with 0.5% HbCO; SLCO, simulated sea level with 4.2% HbCO; and ACO, altitude with 4.2% HbCO. During work at altitude, heart rate (HR), minute ventilation and tidal volume increased and diastolic blood pressures decreased relative to SL. Cardiac output (Qc), stroke volume (SV), and arterial-mixed venous oxygen difference (a--vO2) were similar in smokers and nonsmokers at SL, SLCO, and A. At ACO, nonsmokers increased Qc and SV and decreased a--vO2, but these were not influenced in the smokers. Smokers showed a graded increase in HR when exposed to work in SLCO, A, and ACO. Their lower finger temperatures during A and ACO suggested vasoconstriction in the extremities. Cigarette smokers may be partially adapted to hypoxia.

Influence of ambient temperature on the thermoregulatory responses to 5-hydroxytryptamine, noradrenaline and acetylcholine injected into the lateral cerebral ventricles of sheep, goats and rabbits.

1. The influences of ambient temperature (T(a)) on the thermoregulatory effector activities and the body temperature (T(b)) of intraventricular injections into the sheep, goat and rabbit of 5-hydroxytryptamine (5-HT), noradrenaline (NA), acetylcholine (ACh), carbachol and eserine, have been interpreted in terms of a simple neuronal model of the pathways between thermosensors and thermoregulatory effectors.2. In all three species 5-HT in minimal doses caused a rise in respiratory frequency (RF) and a fall in T(b) at high T(a), and a reduction in EMG activity and a fall in T(b) at low T(a). These effects could be interpreted as those of an excitatory transmitter acting on the warm receptor-heat loss pathway.3. In all three species NA caused a reduction in RF and a rise in T(b) at high T(a), and a reduction in EMG activity and a fall in T(b) at low T(a). These effects are interpreted as those of an inhibitory transmitter acting both on the warm sensor-heat loss pathways and on the cold sensor-heat production pathway.4. The effects of ACh and the cholinomimetic substances carbachol and eserine are complex and more difficult to interpret. In small doses the effects on the sheep and goat are those of an excitatory transmitter on the cold sensor-heat production pathway. There was an increase in EMG activity and a rise in T(b) at low T(a), and a reduction in RF and a rise in T(b) at high T(a). At higher dose levels in the goat and at all dose levels in the rabbit these substances had the reverse effects which are attributed to a synaptic block due to the excess of the excitatory substance.5. The effects of ambient temperature and injected substances upon ear temperature are consistent with the predictions of the model if it is assumed (a) that at high and low ambient temperatures direct thermal effects on ear vessels dominate those of the sympathetic innervation, and (b) that the warm sensor influence is to lower peripheral vasomotor tone, and the cold sensor influence is to increase it.6. The conclusion reached is that when consideration is given to species differences in the thermoneutral ambient temperature and to the possibility that excitatory substances have reversed effects at high dose levels, the effects of 5-HT, NA and ACh in the control of body temperature are very similar in the sheep, goat and rabbit: 5-HT is excitatory on the heat loss pathway, ACh is excitatory on the heat production pathway and NA has an inhibitory influence on both pathways.