Yoon-Sun Kim

Reactive hydroxyl radical-driven oral bacterial inactivation by radio frequency atmospheric plasma

We demonstrated bacterial (Streptococcus mutans) inactivation by a radio frequency power driven atmospheric pressure plasma torch with H2O2 entrained in the feedstock gas. Optical emission spectroscopy identified substantial excited state •OH generation inside the plasma and relative •OH formation was verified by optical absorption. The bacterial inactivation rate increased with increasing •OH generation and reached a maximum 5-log10 reduction with 0.6% H2O2 vapor. Generation of large amounts of toxic ozone is drawback of plasma bacterial inactivation, thus it is significant that the ozone concentration falls within recommended safe allowable levels with addition of H2O2 vapor to the plasma.

Bacterial Inactivation of Wound Infection in a Human Skin Model by Liquid-Phase Discharge Plasma

Background We investigate disinfection of a reconstructed human skin model contaminated with biofilm-formative Staphylococcus aureus employing plasma discharge in liquid. Principal Findings We observed statistically significant 3.83-log10 (p<0.001) and 1.59-log10 (p<0.05) decreases in colony forming units of adherent S. aureus bacteria and 24 h S. aureus biofilm culture with plasma treatment. Plasma treatment was associated with minimal changes in histological morphology and tissue viability determined by means of MTT assay. Spectral analysis of the plasma discharge indicated the presence of highly reactive atomic oxygen radicals (777 nm and 844 nm) and OH bands in the UV region. The contribution of these and other plasma-generated agents and physical conditions to the reduction in bacterial load are discussed. Conclusions These findings demonstrate the potential of liquid plasma treatment as a potential adjunct therapy for chronic wounds.

Reduction in TRPC4 expression specifically attenuates G-protein coupled receptor-stimulated increases in intracellular calcium in human myometrial cells.

Canonical transient receptor potential (TRPC) proteins may play a role in regulating changes in intracellular calcium ([Ca(2+)](i)). Human myometrium expresses TRPC4, TRPC1 and TRPC6 mRNAs in greatest relative abundance. Contributions of TRPC4 to increases in [Ca(2+)](i) were assessed in PHM1-41 and primary human uterine smooth muscle (UtSMC) cells using short hairpin RNAs (shRNAs). Based on a reporter assay screen, one shRNA was selected to construct an adenoviral expression vector (TC4sh1). TC4sh1 induced both mRNA and protein TRPC4 knockdown in PHM1-41 cells without affecting expression of other TRPCs. Signal-regulated Ca(2+) entry (SRCE), defined as a stimulus- and extracellular Ca(2+)-dependent increase in [Ca(2+)](i), was measured in PHM1-41 cells treated with oxytocin (G-protein coupled receptor (GPCR)-stimulated), thapsigargin (store depletion-stimulated), and OAG (diacylglycerol-stimulated), using Fura-2. Cells infected with TC4sh1 exhibited attenuated oxytocin-, ATP- and PGF2alpha-mediated SRCE, but no change in thapsigargin- or OAG-stimulated SRCE. Similar results were obtained in primary uterine smooth muscle cells. Additionally, cells expressing TC4sh1 exhibited a significantly smaller increase in channel activity in response to oxytocin administration than did cells infected with empty virus. These data show that, in human myometrial cells, knockdown of endogenous TRPC4 specifically attenuates GPCR-stimulated, but not thapsigargin- or OAG-stimulated extracellular calcium-dependent increases in [Ca(2+)](i). These data imply that, in this cellular context, the mechanisms regulating extracellular Ca(2+)-dependent increases in [Ca(2+)](i) are differentially affected by different signaling pathways.

Attenuation of canonical transient receptor potential-like channel 6 expression specifically reduces the diacylglycerol-mediated increase in intracellular calcium in human myometrial cells.

An increase in intracellular Ca(2+) ([Ca(2+)](i)) as a result of release of Ca(2+) from intracellular stores or influx of extracellular Ca(2+) contributes to the regulation of smooth muscle contractile activity. Human uterine smooth muscle cells exhibit receptor-, store-, and diacylglycerol (OAG)-mediated extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and express canonical transient receptor potential-like channels (TRPC) mRNAs (predominantly TRPC1, -4, and -6) that have been implicated in SRCE. To determine the role of TRPC6 in human myometrial SRCE, short hairpin RNA constructs were designed that effectively targeted a TRPC6 mRNA reporter for degradation. One sequence was used to produce an adenovirus construct (TC6sh1). TC6sh1 reduced TRPC6 mRNA but not TRPC1, -3, -4, -5, or -7 mRNAs in PHM1-41 myometrial cells. Compared with uninfected cells or cells infected with empty vector, the increase in [Ca(2+)](i) in response to OAG was specifically inhibited by TC6sh1, whereas SRCE responses elicited by either oxytocin or thapsigargin were not changed. Similar findings were observed in primary pregnant human myometrial cells. When PHM1-41 cells were activated by OAG in the absence of extracellular Na(+), the increase in [Ca(2+)](i) was partially reduced. Furthermore, pretreatment with nifedipine, an L-type calcium channel blocker, also partially reduced the OAG-induced [Ca(2+)](i) increase. Similar effects were observed in primary human myometrial cells. These findings suggest that OAG activates channels containing TRPC6 in myometrial cells and that these channels act via both enhanced Na(+) entry coupled to activation of voltage-dependent Ca(2+) entry channels and a nifedipine-independent Ca(2+) entry mechanism to promote elevation of intracellular Ca(2+).

Long Chain Polyunsaturated Fatty Acids Alter Oxytocin Signaling and Receptor Density in Cultured Pregnant Human Myometrial Smooth Muscle Cells

Epidemiological studies and interventional clinical trials indicate that consumption of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) such as docosahexaenoic acid (DHA) lengthen gestational duration. Although the mechanisms are not well understood, prostaglandins (PG) of the 2-series are known to play a role in the initiation and progress of labor. In animal studies, modest DHA provision has been shown to reduce placental and uterine PGE2 and PGF2α, matrix metalloproteinase (MMP)-2 and MMP-9 expression, and placental collagenase activity. However, modulation of PG biosynthesis may not account for all the effects of LC n-3 PUFAs in labor. We investigated one potential PG-independent mechanism of LC PUFA action using cultured pregnant human myometrial smooth muscle cells. Our goal was to characterize the effect of LC PUFA treatment on oxytocin signaling, a potent uterotonic hormone involved in labor. The addition of 10 µM–100 µM DHA or arachidonic acid (AA) to the culture media for 48 h resulted in dose dependent enrichment of these fatty acids in membrane lipid. DHA and AA significantly inhibited phosphatidylinositol turnover and [Ca2+]i mobilization with oxytocin stimulation compared to bovine serum albumin control and equimolar oleic acid. DHA and AA significantly reduced oxytocin receptor membrane concentration without altering binding affinity or rate of receptor internalization. These findings demonstrate a role for LC n-3 PUFAs in regulation of oxytocin signaling and provide new insight into additional mechanisms pertaining to reports of dietary fish and fish oil consumption prolonging gestation.

Changes in Rat Myometrial Plasma Membrane Protein Kinase A Are Confined to Parturition

We have previously shown that pregnant rat myometrial plasma membrane-associated cyclic adenosine monophosphate (cAMP)—dependent protein kinase A (PKA) decreases prior to delivery, coincident with a decline in the inhibitory effect of cAMP on contractant-stimulated parameters. We now find that rat myometrial membrane-associated PKA concentrations in early to mid-pregnancy are equivalent to those in cycling rats. Following the decline associated with parturition, membrane PKA recovers within 1 to 2 days postpartum. Treatment with the antiprogestin onapristone caused a decrease in myometrial membrane PKA catalytic and regulatory subunits compared to untreated controls by 12 hours. This coincided temporally with recently reported increases in electrical and contractile activity. In unilaterally pregnant rats, the decline in plasma membrane PKA was observed in both nonpregnant and pregnant horns but was more rapid in the pregnant horns. These data indicate that the myometrial plasma membrane PKA pattern before and during most of pregnancy is not consistent with progesterone exerting a primary influence on PKA membrane localization. Rather, the fall in membrane PKA associated with parturition may contribute to or be influenced by the increased contractile and electrical activity of labor that is a consequence of the loss of progesterone influence and is not absolutely dependent on the presence of fetuses.