Protamine folds DNA into flowers and loop stacks.

DNA in sperm undergoes an extreme compaction to almost crystalline packing levels. To produce this dense packing, DNA is dramatically reorganized in minutes by protamine proteins. Protamines are positively charged proteins that coat negatively charged DNA and fold it into a series of toroids. The exact mechanism for forming these ∼50-kbp toroids is unknown. Our goal is to study toroid formation by starting at the "bottom" with folding of short lengths of DNA that form loops and working "up" to more folded structures that occur on longer length scales. We previously measured folding of 200-300 bp of DNA into a loop. Here, we look at folding of intermediate DNA lengths (L = 639-3003 bp) that are 2-10 loops long. We observe two folded structures besides loops that we hypothesize are early intermediates in the toroid formation pathway. At low protamine concentrations (∼0.2 µM), we see that the DNA folds into flowers (structures with multiple loops that are positioned so they look like the petals of a flower). Folding at these concentrations condenses the DNA to 25% of its original length, takes seconds, and is made up of many small bending steps. At higher protamine concentrations (≥2 µM), we observe a second folded structure-the loop stack-where loops are stacked vertically one on top of another. These results lead us to propose a two-step process for folding at this length scale: 1) protamine binds to DNA, bending it into loops and flowers, and 2) flowers collapse into loop stacks. These results highlight how protamine uses a bind-and-bend mechanism to rapidly fold DNA, which may be why protamine can fold the entire sperm genome in minutes.

Patient-Specific Risk Factors Associated With the Development of Hyperchloremia in a Neurocritical Care Intensive Care Unit.

BACKGROUND: Hypertonic sodium chloride (HTS) is used in intensive care unit (ICU) settings to manage cerebral edema, intracranial hypertension, and for the treatment of severe hyponatremia. It has been associated with an increased incidence of hyperchloremia; however, there is limited literature focusing on hyperchloremic risk in neurologically injured patients. Objective: The primary objective of this study was to determine risk factors associated with development of hyperchloremia in a neurocritical care (NCC) ICU population. METHODS: This was a retrospective case-control study performed in an adult NCC ICU and included patients receiving HTS. The primary outcome was to evaluate patient characteristics and treatments associated with hyperchloremia. Secondary outcomes included acute kidney injury and mortality. RESULTS: Overall, 133 patients were identified; patients who were hyperchloremic were considered cases (n = 100) and patients without hyperchloremia were considered controls (n = 33). Characteristics and treatments were evaluated with univariate analysis and a logistic regression model. In the multivariate model, APACHE II Score, initial serum osmolality, total 3% saline volume, and total 23.4% saline volume were significant predictors for hyperchloremia. In addition, patients with a serum chloride greater than 113.5 mEq/L were found to have a higher risk of acute kidney injury (AKI) (adjusted OR 3.15; 95% CI 1.10-9.04). CONCLUSIONS: This study demonstrated APACHE II Score, initial serum osmolality, and total 3% and 23.4% saline volumes were associated with developing hyperchloremia in the NCC ICU. In addition, hyperchloremia is associated with an increased risk of AKI.

DNA looping by protamine follows a nonuniform spatial distribution.

DNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription factors like lac repressor or by structural maintenance of chromosomes proteins such as condensin. Here, however, we are interested in a different looping method whereby condensing agents (charge ≥+3) such as protamine proteins neutralize the DNA, causing it to form loops and toroids. We considered two previously proposed mechanisms for DNA looping by protamine. In the first mechanism, protamine stabilizes spontaneous DNA fluctuations, forming randomly distributed loops along the DNA. In the second mechanism, protamine binds and bends the DNA to form a loop, creating a distribution of loops that is biased by protamine binding. To differentiate between these mechanisms, we imaged both spontaneous and protamine-induced loops on short-length (≤1 µm) DNA fragments using atomic force microscopy. We then compared the spatial distribution of the loops to several model distributions. A random looping model, which describes the mechanism of spontaneous DNA folding, fit the distribution of spontaneous loops, but it did not fit the distribution of protamine-induced loops. Specifically, it failed to predict a peak in the spatial distribution of loops at an intermediate location along the DNA. An electrostatic multibinding model, which was created to mimic the bind-and-bend mechanism of protamine, was a better fit of the distribution of protamine-induced loops. In this model, multiple protamines bind to the DNA electrostatically within a particular region along the DNA to coordinate the formation of a loop. We speculate that these findings will impact our understanding of protamine's in vivo role for looping DNA into toroids and the mechanism of DNA condensation by condensing agents more broadly.

Protamine loops DNA in multiple steps.

Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA into a single loop. Current models for DNA loop formation are one-step, all-or-nothing models with a looped state and an unlooped state. However, when we use a Tethered Particle Motion (TPM) assay to measure the dynamic, real-time looping of DNA by protamine, we observe the presence of multiple folded states that are long-lived (∼100 s) and reversible. In addition, we measure folding on DNA molecules that are too short to form loops. This suggests that protamine is using a multi-step process to loop the DNA rather than a one-step process. To visualize the DNA structures, we used an Atomic Force Microscopy (AFM) assay. We see that some folded DNA molecules are loops with a ∼10-nm radius and some of the folded molecules are partial loops-c-shapes or s-shapes-that have a radius of curvature of ∼10 nm. Further analysis of these structures suggest that protamine is bending the DNA to achieve this curvature rather than increasing the flexibility of the DNA. We therefore conclude that protamine loops DNA in multiple steps, bending it into a loop.

Intensity-dependent timing and precision of startle response latency in larval zebrafish.

KEY POINTS: Using high-speed videos time-locked with whole-animal electrical recordings, simultaneous measurement of behavioural kinematics and field potential parameters of C-start startle responses allowed for discrimination between short-latency and long-latency C-starts (SLCs vs. LLCs) in larval zebrafish. Apart from their latencies, SLC kinematics and SLC field potential parameters were intensity independent. Increasing stimulus intensity increased the probability of evoking an SLC and decreased mean SLC latencies while increasing their precision; subtraction of field potential latencies from SLC latencies revealed a fixed time delay between the two measurements that was intensity independent. The latency and the precision in the latency of the SLC field potentials were linearly correlated to the latencies and precision of the first evoked action potentials (spikes) in hair-cell afferent neurons of the lateral line. Together, these findings indicate that first spike latency (FSL) is a fast encoding mechanism that can serve to precisely initiate startle responses when speed is critical for survival. ABSTRACT: Vertebrates rely on fast sensory encoding for rapid and precise initiation of startle responses. In afferent sensory neurons, trains of action potentials (spikes) encode stimulus intensity within the onset time of the first evoked spike (first spike latency; FSL) and the number of evoked spikes. For speed of initiation of startle responses, FSL would be the more advantageous mechanism to encode the intensity of a threat. However, the intensity dependence of FSL and spike number and whether either determines the precision of startle response initiation is not known. Here, we examined short-latency startle responses (SLCs) in larval zebrafish and tested the hypothesis that first spike latencies and their precision (jitter) determine the onset time and precision of SLCs. We evoked startle responses via activation of Channelrhodopsin (ChR2) expressed in ear and lateral line hair cells and acquired high-speed videos of head-fixed larvae while simultaneously recording underlying field potentials. This method allowed for discrimination between primary SLCs and less frequent, long-latency startle responses (LLCs). Quantification of SLC kinematics and field potential parameters revealed that, apart from their latencies, they were intensity independent. We found that increasing stimulus intensity decreased SLC latencies while increasing their precision, which was significantly correlated with corresponding changes in field potential latencies and their precision. Single afferent neuron recordings from the lateral line revealed a similar intensity-dependent decrease in first spike latencies and their jitter, which could account for the intensity-dependent changes in timing and precision of startle response latencies.

A Surface-Coupled Optical Trap with 1-bp Precision via Active Stabilization.

Optical traps can measure bead motions with Å-scale precision. However, using this level of precision to infer 1-bp motion of molecular motors along DNA is difficult, since a variety of noise sources degrade instrumental stability. In this chapter, we detail how to improve instrumental stability by (1) minimizing laser pointing, mode, polarization, and intensity noise using an acousto-optical-modulator mediated feedback loop and (2) minimizing sample motion relative to the optical trap using a three-axis piezo-electric-stage mediated feedback loop. These active techniques play a critical role in achieving a surface stability of 1 Å in 3D over tens of seconds and a 1-bp stability and precision in a surface-coupled optical trap over a broad bandwidth (Δf = 0.03-2 Hz) at low force (6 pN). These active stabilization techniques can also aid other biophysical assays that would benefit from improved laser stability and/or Å-scale sample stability, such as atomic force microscopy and super-resolution imaging.

Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD-DNA complexes.

RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 µM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5' strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket.

Sensorimotor structure of Drosophila larva phototaxis.

The avoidance of light by fly larvae is a classic paradigm for sensorimotor behavior. Here, we use behavioral assays and video microscopy to quantify the sensorimotor structure of phototaxis using the Drosophila larva. Larval locomotion is composed of sequences of runs (periods of forward movement) that are interrupted by abrupt turns, during which the larva pauses and sweeps its head back and forth, probing local light information to determine the direction of the successive run. All phototactic responses are mediated by the same set of sensorimotor transformations that require temporal processing of sensory inputs. Through functional imaging and genetic inactivation of specific neurons downstream of the sensory periphery, we have begun to map these sensorimotor circuits into the larval central brain. We find that specific sensorimotor pathways that govern distinct light-evoked responses begin to segregate at the first relay after the photosensory neurons.

Back-scattered detection yields viable signals in many conditions.

Precision position-sensing is required for many microscopy techniques. One promising method, back-scattered detection (BSD), is incredibly sensitive, allowing for position measurements at the level of tens of picometers in three dimensions. In BSD the position of a micron-sized bead is measured by back-scattering a focused laser beam off the bead and imaging the resulting interference pattern onto a detector. Since the detection system geometry is confined to one side of the objective, the technique is compatible with platforms that have restricted optical access (e.g. magnetic tweezers, atomic force microscopy, and microfluidics). However, general adoption of BSD may be limited according to a recent theory [Volpe et al., J. Appl. Phys. 102, 084701, 2007] that predicts diminished signals under certain conditions. We directly measured the BSD response while varying the experimental conditions, including bead radius, numerical aperture, and relative index. Contrary to the proposed theory, we find that all experimental conditions tested produced a viable signal for atomic-scale measurements.

Ultrastable atomic force microscopy: atomic-scale stability and registration in ambient conditions.

Instrumental drift in atomic force microscopy (AFM) remains a critical, largely unaddressed issue that limits tip-sample stability, registration, and the signal-to-noise ratio during imaging. By scattering a laser off the apex of a commercial AFM tip, we locally measured and thereby actively controlled its three-dimensional position above a sample surface to <40 pm (Deltaf = 0.01-10 Hz) in air at room temperature. With this enhanced stability, we overcame the traditional need to scan rapidly while imaging and achieved a 5-fold increase in the image signal-to-noise ratio. Finally, we demonstrated atomic-scale ( approximately 100 pm) tip-sample stability and registration over tens of minutes with a series of AFM images on transparent substrates. The stabilization technique requires low laser power (<1 mW), imparts a minimal perturbation upon the cantilever, and is independent of the tip-sample interaction. This work extends atomic-scale tip-sample control, previously restricted to cryogenic temperatures and ultrahigh vacuum, to a wide range of perturbative operating environments.

Precision surface-coupled optical-trapping assay with one-basepair resolution.

The most commonly used optical-trapping assays are coupled to surfaces, yet such assays lack atomic-scale ( approximately 0.1 nm) spatial resolution due to drift between the surface and trap. We used active stabilization techniques to minimize surface motion to 0.1 nm in three dimensions and decrease multiple types of trap laser noise (pointing, intensity, mode, and polarization). As a result, we achieved nearly the thermal limit (<0.05 nm) of bead detection over a broad range of trap stiffness (k(T) = 0.05-0.5 pN/nm) and frequency (Deltaf = 0.03-100 Hz). We next demonstrated sensitivity to one-basepair (0.34-nm) steps along DNA in a surface-coupled assay at moderate force (6 pN). Moreover, basepair stability was achieved immediately after substantial (3.4 pN) changes in force. Active intensity stabilization also led to enhanced force precision ( approximately 0.01%) that resolved 0.1-pN force-induced changes in DNA hairpin unfolding dynamics. This work brings the benefit of atomic-scale resolution, currently limited to dual-beam trapping assays, along with enhanced force precision to the widely used, surface-coupled optical-trapping assay.

Stabilization of an optical microscope to 0.1 nm in three dimensions.

Mechanical drift is a long-standing problem in optical microscopy that occurs in all three dimensions. This drift increasingly limits the resolution of advanced surface-coupled, single-molecule experiments. We overcame this drift and achieved atomic-scale stabilization (0.1 nm) of an optical microscope in 3D. This was accomplished by measuring the position of a fiducial mark coupled to the microscope cover slip using back-focal-plane (BFP) detection and correcting for the drift using a piezoelectric stage. Several significant factors contributed to this experimental realization, including (i) dramatically reducing the low frequency noise in BFP detection, (ii) increasing the sensitivity of BFP detection to vertical motion, and (iii) fabricating a regular array of nanometer-sized fiducial marks that were firmly coupled to the cover slip. With these improvements, we achieved short-term (1 s) stabilities of 0.11, 0.10, and 0.09 nm (rms) and long-term (100 s) stabilities of 0.17, 0.12, and 0.35 nm (rms) in x, y, and z, respectively, as measured by an independent detection laser.

Back-scattered detection provides atomic-scale localization precision, stability, and registration in 3D.

State-of-the-art microscopy techniques (e.g., atomic force microscopy, scanning-tunneling microscopy, and optical tweezers) are sensitive to atomic-scale (100 pm) displacements. Yet, sample drift limits the ultimate potential of many of these techniques. We demonstrate a general solution for sample control in 3D using back-scattered detection (BSD) in both air and water. BSD off a silicon disk fabricated on a cover slip enabled 19 pm lateral localization precision (Deltaf = 0.1-50 Hz) with low crosstalk between axes (