College Years Spencer Lee

From his early years in South Korea, to becoming the CEO of the largest plumbing company in North America, Spencer Lee has accomplished what many would call the American Dream. His hard but balanced work ethic, along with his motivation and positive outlook, are just some of Lee\u27s attributes that have taken him to where he is today. Find out about his college years, along with some of his more personal insights, below

Efficient preparation of internally modified single-molecule constructs using nicking enzymes

Investigations of enzymes involved in DNA metabolism have strongly benefited from the establishment of single molecule techniques. These experiments frequently require elaborate DNA substrates, which carry chemical labels or nucleic acid tertiary structures. Preparing such constructs often represents a technical challenge: long modified DNA molecules are usually produced via multi-step processes, involving low efficiency intermolecular ligations of several fragments. Here, we show how long stretches of DNA (>50 bp) can be modified using nicking enzymes to produce complex DNA constructs. Multiple different chemical and structural modifications can be placed internally along DNA, in a specific and precise manner. Furthermore, the nicks created can be resealed efficiently yielding intact molecules, whose mechanical properties are preserved. Additionally, the same strategy is applied to obtain long single-strand overhangs subsequently used for efficient ligation of ss- to dsDNA molecules. This technique offers promise for a wide range of applications, in particular single-molecule experiments, where frequently multiple internal DNA modifications are required

Sequential action of ATPase, ATP, ADP, Pi and dsDNA in procapsid-free system to enlighten mechanism in viral dsDNA packaging

Many cells and double-stranded DNA (dsDNA) viruses contain an AAA+ ATPase that assembles into oligomers, often hexamers, with a central channel. The dsDNA packaging motor of bacteriophage phi29 also contains an ATPase to translocate dsDNA through a dodecameric channel. The motor ATPase has been investigated substantially in the context of the entire procapsid. Here, we report the sequential action between the ATPase and additional motor components. It is suggested that the contact of ATPase to ATP resulted in its conformational change to a higher binding affinity toward dsDNA. It was found that ATP hydrolysis led to the departure of dsDNA from the ATPase/dsDNA complex, an action that is speculated to push dsDNA to pass the connector channel. Our results suggest that dsDNA packaging goes through a combined effort of both the gp16 ATPase for pushing and the channel as a one-way valve to control the dsDNA translocation direction. Many packaging models have previously been proposed, and the packaging mechanism has been contingent upon the number of nucleotides packaged per ATP relative to the 10.5 bp per helical turn for B-type dsDNA. Both 2 and 2.5 bp per ATP have been used to argue for four, five or six discrete steps of dsDNA translocation. Combination of the two distinct roles of gp16 and connector renews the perception of previous dsDNA packaging energy calculations and provides insight into the discrepancy between 2 and 2.5 bp per ATP

Graphical models for inferring single molecule dynamics

<p>Abstract</p> <p>Background</p> <p>The recent explosion of experimental techniques in single molecule biophysics has generated a variety of novel time series data requiring equally novel computational tools for analysis and inference. This article describes in general terms how graphical modeling may be used to learn from biophysical time series data using the variational Bayesian expectation maximization algorithm (VBEM). The discussion is illustrated by the example of single-molecule fluorescence resonance energy transfer (smFRET)<it> versus</it> time data, where the smFRET time series is modeled as a hidden Markov model (HMM) with Gaussian observables. A detailed description of smFRET is provided as well.</p> <p>Results</p> <p>The VBEM algorithm returns the model’s evidence and an approximating posterior parameter distribution given the data. The former provides a metric for model selection via maximum evidence (ME), and the latter a description of the model’s parameters learned from the data. ME/VBEM provide several advantages over the more commonly used approach of maximum likelihood (ML) optimized by the expectation maximization (EM) algorithm, the most important being a natural form of model selection and a well-posed (non-divergent) optimization problem.</p> <p>Conclusions</p> <p>The results demonstrate the utility of graphical modeling for inference of dynamic processes in single molecule biophysics.</p

Single molecule studies of a viral DNA packaging motor

Protein complexes belonging to the super-family of ATPases Associated with diverse Activities of the Cell (AAA+), form rings that function as molecular motors to translocate DNA during important processes in the cell, including replication, recombination, transposition of genes, conjugation, genome segregation and viral DNA packaging. We have carried out studies of the DNA packaging motor of bacteriophage Ф29, a motor with five ATPase subunits, directed at understanding the mechanism of force generation. Using optical-tweezers-based single molecule assays, we applied precise forces to these motors and followed the packaging dynamics. First, we characterized the mechano-chemistry of the motor, how the chemical hydrolysis cycle of ATP couples to the mechanical cycle of pumping DNA. We studied the dependence of the motor's dynamics in response to a range of forces and chemical potentials. We have shown that the power stroke of the motor does not occur during the ATP binding, but during the release of phosphate. Next, by the use of non-hydrolysable ATP analogs, we showed that the firing of the motor-subunits is highly coordinated. Our findings allow us to postulate the first mechano-chemical scheme for this motor. We then studied the nature of the interactions that allow the motor to bind and translocate DNA. Using synthetic DNA substrates that differed either in charge, chemical structure, or continuity, we probed the importance of various specific interactions. We found that the motor makes periodic contacts with the DNA backbone every 10 bp, tracking the 5'-3' strand but not the 3'-5' strand. The motor packaged actively against an applied force an un-charged substrate better than an abasic substrate. We also observe that the motor can translocate through large discontinuities or bulges in the DNA. We conclude that the motor likely holds and orients the DNA via ionic-interactions but sterically pushes on the DNA mostly via the bases using a non-specific 'friction drive'. Our findings for the mechanism of force generation of the bacteriophage Ф29 DNA packaging motor constitute a significant advance in the understanding of the AAA+ DNA translocases

General surgical unit experience of traumatic vascular injuries in an Australian regional hospital

Background: Vascular trauma is a significant burden to the regional community due to the associated high morbidity and mortality from the complications of the injuries. This may be due to the complexity of the injuries and the issues with initial management at the regional hospitals. Vascular injury management training should improve the outcome of initial surgical management by the general surgical team in regional centres. The study aims to describe the epidemiology and management of vascular injury in regional hospitals in Queensland, Australia.Methods: A retrospective descriptive analysis was performed using the data of patients with traumatic vascular injuries between January 2017 and July 2021 presented to Hervey Bay hospital in Queensland, Australia.Results: Fifty-nine patients with vascular injuries were reviewed. The reported number of penetrating and blunt trauma cases were 43 (72.9%) and 16 (27.1%) respectively, and 42 (71.2%) were males. Most mechanisms of injury were cuts (n=32, 54.2%), followed by falls (n=16, 27.1%) and stabs (n=11, 18.7%). Fifty-one (86.4%) were initially surgically managed regionally, with 21 (35.6%) requiring transfer to a tertiary trauma centre. Outcomes of the incidents resulted in one (1.7%) patient requiring amputation, three (5.1%) needing a fasciotomy and the mortality of two (3.4%) patients.Conclusions: Vascular trauma causes a significant burden to Australian regional hospitals. Identifying injury patterns and common causes for vascular injury will help in early identification and prompt management. Vascular trauma management training should improve the quality of care from the general surgeons in the regional centres.</jats:p

Overprescribing proton pump inhibitors

Is expensive and not evidence base

THE INFLUENCE OF MANUFACTURING PROCESSES AND OPTICAL MEASUREMENT METHODS ON THE DAMAGE BEHAVIOR OF HX340LAD MICRO-ALLOYED STEELS

This study deals with the damage behavior of metallic materials by the application of different manufacturing processes and using different optical measurement methods to identify the crack initiation in the damage specimen. The study is intended to highlight the importance of considering manufacturing processes and optical measurement methods in a numerical simulation when analyzing the damage behavior of metallic materials. To describe the damage behavior of the material in the process chain simulations, it is important to calibrate the parameters of damage model more accurately. These parameters are determined using experimental investigation of desired damage specimens. In this regard, a selected damage specimen manufactured by different cutting processes is first experimentally and then numerically investigated. It is shown that the manufacturing process and the optical measurement methods influence the stress state analyzed in the numerical simulation.</jats:p