Skeletal muscle regeneration and the role of Sirtuin 1

The main objective of this master\u27s thesis project was to elucidate the role of the deacetylase Sirtuin 1 (Sirt1) in skeletal muscle regeneration. We developed two specific aims to accomplish this task, which involved separate studies in order to study the effects of Sirt1 in multiple models of regeneration. The results from these studies may potentially lead to the development of novel therapies to treat various clinical conditions which require muscle regeneration.;Specific aim 1 utilized resveratrol, a Sirt1 activator, as a supplement to improve muscle regeneration following a period of disuse in aged animals. Aged Fisher 344 x Brown Norway rats were hindlimb suspended for a total of fourteen days (HS), whereupon one-half of the animals were sacrificed and the remainder were unloaded and allowed a fourteen day recovery period of normal ambulation (R), at which point they were sacrificed. During the experimental period, each rat received either 1ml of resveratrol (125 mg/kg) or 1ml of carboxymethylcellulose dissolved in distilled water (vehicle) via oral gavage at approximately the same time each morning. Additionally, a group of vehicle treated animals maintained normal ambulation throughout the protocol and served as cage controls for the suspended animals.;Resveratrol was not able to attenuate losses in either body or muscle wet weights following HS and did not provide any additional benefits to these measures during the R period. Surprisingly, resveratrol treated animals did not fare as well on a fatigue protocol and few differences were observed upon examination of a force-frequency curve. However, fiber type analyses revealed a shift towards increased type IIA myosin heavy chain (MyHC) fibers following HS. Resveratrol also attenuated decreases in fiber area in type IIA MyHC fibers following HS and in both type IIA and IIB MyHC fibers following the R period.;Our results from this study indicate a potential for the use of resveratrol in benefitting skeletal muscle regeneration in aged populations following periods of disuse such as extended hospitalizations. Although resveratrol was unable to maintain muscle function as determined by the force characteristics, it clearly benefitted muscle fiber size which is important in this population as the elderly have diminished regenerative abilities. Future research should be aimed at determining the optimal dosage to fully maximize the benefits of this supplement in skeletal muscle regeneration.;Specific aim 2 attempted to establish the role of Sirt1 in muscle regeneration using transgenic mice which either overexpressed (Sirt1+/+) or had diminished Sirt1 expression (Sirt1-/-) in the skeletal muscles. Tibialis anterior (TA) muscles were examined either 3 or 7 days following a cardiotoxin (CTX) injection in the animals left TA muscles. The right TA muscles were injected with sterile PBS and were used as intranimal controls for the CTX administration.;Increasing Sirt1 expression did not have an effect on muscle mass or fiber area at either 3 or 7 days. However, increasing its expression attenuated decreases in maximal tetanic force production at both 3 and 7 days post-injury while there were no differences observed in these forces in the Sirt1 -/- animals. There were no differences observed between any of the groups in BrdU incorporation and furthermore, in apoptosis as determined via TUNEL staining.;Results from this study indicate that Sirt1 may not play a significant role in muscle mass or fiber area during the early stages of muscle regeneration. However, increasing Sirt1 expression was beneficial in that it improved muscle force characteristics which would suggest improved functional recovery in these animals. Therefore, although inconclusive, increasing Sirt1 expression may be beneficial at improving skeletal muscle regeneration in that it may reduce functional impairments in muscle viability. Future research may thus be directed at developing therapies to exploit these benefits and be applied toward clinical populations which require skeletal muscle regeneration

Spline Based Intrusion Detection in Vehicular Ad Hoc Networks (VANET)

Intrusion detection systems (IDSs) play a crucial role in the identification and mitigation for attacks on host systems. Of these systems, vehicular ad hoc networks (VANETs) are particularly difficult to protect due to the dynamic nature of their clients and their necessity for constant interaction with their respective cyber-physical systems. Currently, there is a need for a VANET-specific IDS that can satisfy these requirements. Spline function-based IDSs have shown to be effective in traditional network settings. By examining the various construction of splines and testing their robustness, the viability for a spline-based IDS can be determined

Effects of Resveratrol on the Recovery of Muscle Mass Following Disuse in the Plantaris Muscle of Aged Rats

Aging is associated with poor skeletal muscle regenerative ability following extended periods of hospitalization and other forms of muscular disuse. Resveratrol (3,5,4’-trihydroxystilbene) is a natural phytoalexin which has been shown in skeletal muscle to improve oxidative stress levels in muscles of aged rats. As muscle disuse and reloading after disuse increases oxidative stress, we hypothesized that resveratrol supplementation would improve muscle regeneration after disuse. A total of thirty-six male Fisher 344 × Brown Norway rats (32 mo.) were treated with either a water vehicle or resveratrol via oral gavage. The animals received hindlimb suspension for 14 days. Thereafter, they were either sacrificed or allowed an additional 14 day period of cage ambulation during reloading. A total of six rats from the vehicle and the resveratrol treated groups were used for the hindlimb suspension and recovery protocols. Furthermore, two groups of 6 vehicle treated animals maintained normal ambulation throughout the experiment, and were used as control animals for the hindlimb suspension and reloading groups. The data show that resveratrol supplementation was unable to attenuate the decreases in plantaris muscle wet weight during hindlimb suspension but it improved muscle mass during reloading after hindlimb suspension. Although resveratrol did not prevent fiber atrophy during the period of disuse, it increased the fiber cross sectional area of type IIA and IIB fibers in response to reloading after hindlimb suspension. There was a modest enhancement of myogenic precursor cell proliferation in resveratrol-treated muscles after reloading, but this failed to reach statistical significance. The resveratrol-associated improvement in type II fiber size and muscle mass recovery after disuse may have been due to decreases in the abundance of pro-apoptotic proteins Bax, cleaved caspase 3 and cleaved caspase 9 in reloaded muscles. Resveratrol appears to have modest therapeutic benefits for improving muscle mass after disuse in aging

Fatigue Behavior of Welded Connections Enhanced with UIT and Bolting

A common problem in bridges employing welded steel girders is development of fatigue cracks at the ends of girder coverplates. Fatigue cracks tend to form at the toes of the transverse welds connecting a coverplate to a girder flange since this detail has a region of very high stress concentration. Because many aging bridges employ these fatigue-prone, AASHTO fatigue Category E or E' details, a means to effectively enhance the fatigue lives of these details is being sought. A research project funded by the Kansas Department of Transportation was undertaken at the University of Kansas to investigate the fatigue life enhancement afforded by two retrofit methods. One retrofit method was similar to a method described in the AASTHO Bridge Design Specification (AASHTO 2004) and involved pretensioned bolts being added to the ends of coverplates near the transverse welds. Unlike the AASHTO bolting procedure, the modified bolting procedure studied during this project utilized coverplates having transverse fillet welds that were left in the as-fabricated state. The other retrofit method was the use of a proprietary needle peening procedure called Ultrasonic Impact Treatment (UIT). Results of the research project showed that UIT was highly effective at enhancing the fatigue lives of coverplate end details while the bolting procedure was ineffective. Weld treatment with UIT resulted in an improvement in fatigue life over control specimens by a factor of 25. This translated in an improvement from an AASTHO fatigue Category E detail rating to and AASHTO fatigue Category A detail rating. The modified coverplate bolting procedure tested during this project had either no effect on fatigue life or, in some cases, had a detrimental effect on fatigue life. The coverplate bolting procedure included in the AASHTO specification allows a coverplate end detail to achieve a fatigue Category B resistance when bolted rather than transversely welded. 11 Therefore, the modified bolting procedure tested during this project was much less effective at enhancing fatigue life than either the AASHTO bolting procedure or UIT

Band Gap Engineering with Ultralarge Biaxial Strains in Suspended Monolayer MoS2

We demonstrate the continuous and reversible tuning of the optical band gap of suspended monolayer MoS2 membranes by as much as 500 meV by applying very large biaxial strains. By using chemical vapor deposition (CVD) to grow crystals that are highly impermeable to gas, we are able to apply a pressure difference across suspended membranes to induce biaxial strains. We observe the effect of strain on the energy and intensity of the peaks in the photoluminescence (PL) spectrum, and find a linear tuning rate of the optical band gap of 99 meV/%. This method is then used to study the PL spectra of bilayer and trilayer devices under strain, and to find the shift rates and Gr\"uneisen parameters of two Raman modes in monolayer MoS2. Finally, we use this result to show that we can apply biaxial strains as large as 5.6% across micron sized areas, and report evidence for the strain tuning of higher level optical transitions.Comment: Nano Lett., Article ASA

The full genome sequence of three strains of Jamestown Canyon virus and their pathogenesis in mice or monkeys

<p>Abstract</p> <p>Background</p> <p>Jamestown Canyon virus (JCV), family <it>Bunyaviridae</it>, is a mosquito-borne pathogen endemic in the United States and Canada that can cause encephalitis in humans and is considered an emerging threat to public health. The virus is genetically similar to Inkoo virus circulating in Europe, suggesting that much of the northern hemisphere contains JCV or similar variants.</p> <p>Results</p> <p>We have completed the sequence of three isolates of JCV collected in geographically diverse locations over a 57 year time span. The nucleotide identity for the three strains is 90, 83, and 85% for the S, M, and L segments respectively whereas the percent identify for the predicted amino acid sequences of the N, NS_S, M poly, G_N, NS_M, G_C, and L proteins was 97, 91, 94, 98, 91, 94, and 97%, respectively. In Swiss Webster mice, each JCV isolate exhibits low neuroinvasiveness but high infectivity. Two of the three JCV isolates were highly neurovirulent after IC inoculation whereas one isolate, JCV/03/CT, exhibited low neurovirulence. In rhesus monkeys, JCV infection is accompanied by a low-titered viremia, lack of clinical disease, but a robust neutralizing antibody response.</p> <p>Conclusions</p> <p>The first complete sequence of JCV is reported for three separate isolates, and a relatively high level of amino acid sequence conservation was observed even for viruses isolated 57 years apart indicating that the virus is in relative evolutionary stasis. JCV is highly infectious for mice and monkeys, and these animals, especially mice, represent useful experimental hosts for further study.</p

Correlations between the Electronic Properties of \u3cem\u3eShewanella oneidensis\u3c/em\u3e Cytochrome \u3cem\u3ec\u3c/em\u3e Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV–visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV–vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV–vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site’s midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV–vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme’s histidine axial ligands was replaced with a methionine

Genome sequence analysis of La Crosse virus and in vitro and in vivo phenotypes

<p>Abstract</p> <p>Background</p> <p>La Crosse virus (LACV), family <it>Bunyaviridae</it>, is a mosquito-borne virus recognized as a major cause of pediatric encephalitis in North America with 70–130 symptomatic cases each year. The virus was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl who suffered encephalitis and died in La Crosse, Wisconsin. The majority of LACV infections are mild and never reported, however, serologic studies estimate infection rates of 10–30/100,000 in endemic areas.</p> <p>Results</p> <p>In the present study, sequence analysis of the complete LACV genomes of low-passage LACV/human/1960, LACV/mosquito/1978, and LACV/human/1978 strains and of biologically cloned derivatives of each strain, indicates that circulating LACVs are genetically stable over time and geographic distance with 99.6–100%, 98.9–100%, 97.8–99.6%, and 99.2–99.7% amino acid identity for N, NsS, M polyprotein, and L proteins respectively. We identified 5 amino acid differences in the RNA polymerase and 4 nucleotide differences in the non-coding region of the L segment specific to the human virus isolates, which may result in altered disease outcomes.</p> <p>Conclusion</p> <p>All three wild type viruses had similar <it>in vitro </it>growth kinetics and phenotypes in mosquito C6/36 and Vero cells, and similar levels of neurovirulence and neuroinvasiveness in Swiss Webster mice. The biologically cloned derivative of LACV/human/1960 was significantly less neuroinvasive than its uncloned parent and differed in sequence at one amino acid position in the G_Nglycoprotein, identifying this residue as an attenuating mutation.</p

Inhibition of the \u3cem\u3edapE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Succinyl- ʟ, ʟ-diaminopimelic Acid Desuccinylase from \u3cem\u3eNeisseria meningitidis\u3c/em\u3e by ʟ-Captopril

Binding of the competitive inhibitor ʟ-captopril to the dapE-encoded N-succinyl-ʟ, ʟ-diaminopimelic acid desuccinylase from Neisseria meningitidis (NmDapE) was examined by kinetic, spectroscopic, and crystallographic methods. ʟ-Captopril, an angiotensin-converting enzyme (ACE) inhibitor, was previously shown to be a potent inhibitor of the DapE from Haemophilus influenzae (HiDapE) with an IC50 of 3.3 μM and a measured Ki of 1.8 μM and displayed a dose-responsive antibiotic activity toward Escherichia coli. ʟ-Captopril is also a competitive inhibitor of NmDapE with a Ki of 2.8 μM. To examine the nature of the interaction of ʟ-captopril with the dinuclear active site of DapE, we have obtained electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) data for the enzymatically hyperactive Co(II)-substituted forms of both HiDapE and NmDapE. EPR and MCD data indicate that the two Co(II) ions in DapE are antiferromagnetically coupled, yielding an S = 0 ground state, and suggest a thiolate bridge between the two metal ions. Verification of a thiolate-bridged dinuclear complex was obtained by determining the three-dimensional X-ray crystal structure of NmDapE in complex with ʟ-captopril at 1.8 Å resolution. Combination of these data provides new insights into binding of ʟ-captopril to the active site of DapE enzymes as well as important inhibitor–active site residue interaction’s. Such information is critical for the design of new, potent inhibitors of DapE enzymes

Late Miocene-Quaternary fault evolution and interaction in the southern California Inner Continental Borderland

Changing conditions along plate boundaries are thought to result in the reactivation of preexisting structures. The offshore southern California Borderland has undergone dramatic adjustments as conditions changed from subduction tectonics to transform tectonics, including major Miocene oblique extension, followed by transpressional fault reactivation. However, consensus is still lacking about stratigraphic age models, fault geometry, and slip history for the near-offshore area between southern Los Angeles and San Diego (California, USA). We interpret an extensive data set of seismic reflection, bathymetric, and stratigraphic data from that area to determine the three-dimensional geometry and kinematic evolution of the faults and folds and document how preexisting structures have changed their activity and type of slip through time. The resulting structural representation reveals a moderately landward-dipping San Mateo–Carlsbad fault that converges downward with the steeper, right-lateral Newport-Inglewood fault, forming a fault wedge affected by Quaternary contractional folding. This fault wedge deformed in transtension during late Miocene through Pliocene time. Subsequently, the San Mateo–Carlsbad fault underwent 0.6–1.0 km displacement, spatially varying between reverse right lateral and transtensional right lateral. In contrast, shallow parts of the previously identified gently dipping Oceanside detachment and the faults above it appear to have been inactive since the early Pliocene. These observations, together with new and revised geometric representations of additional steeper faults, and the evidence for a pervasive strike-slip component on these nearshore faults, suggest a need to revise the earthquake hazard estimates for the coastal region