The Evolving Role of Dental Responders on Interprofessional Emergency Response Teams.

Disaster and pandemic response events require an interprofessional team of health care responders to organize and work together in high-pressure, time-critical situations. Civilian oral health care professionals have traditionally been limited to forensic identification of human remains. However, after the bombing of the Twin Towers in New York, federal agencies realized that dentists can play significant roles in disaster and immunization response, especially on interprofessional responder teams. Several states have begun to incorporate dentists into the first responder community. This article discusses the roles of dental responders and highlights legislative advancements and advocacy efforts supporting the dental responder.

Just-in-time training of dental responders in a simulated pandemic immunization response exercise.

ABSTRACT OBJECTIVE: The reauthorization of the Pandemic and All-Hazards Preparedness Act in 2013 incorporated the dental profession and dental professionals into the federal legislation governing public health response to pandemics and all-hazard situations. Work is now necessary to expand the processes needed to incorporate and train oral health care professionals into pandemic and all-hazard response events. METHODS: A just-in-time (JIT) training exercise and immunization drill using an ex vivo porcine model system was conducted to demonstrate the rapidity to which dental professionals can respond to a pandemic influenza scenario. Medical history documentation, vaccination procedures, and patient throughput and error rates of 15 dental responders were evaluated by trained nursing staff and emergency response personnel. RESULTS: The average throughput (22.33/hr) and medical error rates (7 of 335; 2.08%) of the dental responders were similar to those found in analogous influenza mass vaccination clinics previously conducted using certified public health nurses. CONCLUSIONS: The dental responder immunization drill validated the capacity and capability of dental professionals to function as a valuable immunization resource. The ex vivo porcine model system used for JIT training can serve as a simple and inexpensive training tool to update pandemic responders' immunization techniques and procedures supporting inoculation protocols.

Part I-mechanism of adaptation: high nitric oxide adapted A549 cells show enhanced DNA damage response and activation of antiapoptotic pathways.

Our previous studies demonstrate that A549, a human lung adenocarcinoma line, could be adapted to the free radical nitric oxide (NO([Symbol: see text])). NO([Symbol: see text]) has been shown to be overexpressed in human tumors. The original cell line, A549 (parent), and the newly adapted A549-HNO (which has a more aggressive phenotype) serves as a useful model system to study the role of NO([Symbol: see text]) in tumor biology. It is well known that DNA damage response (DDR) is altered in cancer cells and NO([Symbol: see text]) is known to cause DNA damage. Modulations in molecular mechanisms involved in DNA damage response in A549-HNO cells can provide better insights into the enhanced growth behavior of these cells. Thus, here, we carried out a series of time course experiments by treating A549 and A549-HNO cells with NO([Symbol: see text]) donor and examining levels of proteins involved in the DDR pathway. We observed induced expression of key components of DDR pathway in A549-HNO cells. The HNO cells showed sustained expression of key proteins involved in both nonhomologous end joining (NHEJ) and homologous recombination pathways, whereas parent cells only expressed low levels of NHEJ pathway proteins. Further with prolonged NO([Symbol: see text]) exposure, ATR, Chk1, and p53 were activated and upregulated in HNO cells. Activation of p53 results in inhibition of apoptosis through induced Mcl1 expression. It also leads to cell cycle modulation. Interestingly, several reports show that cancer stem cells have enhanced expression of proteins involved in DNA damage response and also activated an antiapoptotic response. Our results here suggest that our HNO adapted A549 cells have increased activation of DNA damage response pathway proteins which can lead to better DNA repair function. Enhanced DDR leads to activation of antiapoptosis response and modulation in the cell cycle which may lead to better survival of these cells under harsh conditions. Thus, our present investigation further supports the hypothesis that HNO exposure leads to survival of these cells.

Photodynamic therapy: occupational hazards and preventative recommendations for clinical administration by healthcare providers.

OBJECTIVE: Photodynamic therapy (PDT) as a medical treatment for cancers is an increasing practice in clinical settings, as new photosensitizing chemicals and light source technologies are developed and applied. PDT involves dosing patients with photosensitizing drugs, and then exposing them to light using a directed energy device in order to manifest a therapeutic effect. Healthcare professionals providing PDT should be aware of potential occupational health and safety hazards posed by these treatment devices and photosensitizing agents administered to patients. MATERIALS AND METHODS: Here we outline and identify pertinent health and safety considerations to be taken by healthcare staff during PDT procedures. RESULTS: Physical hazards (for example, non-ionizing radiation generated by the light-emitting device, with potential for skin and eye exposure) and chemical hazards (including the photosensitizing agents administered to patients that have the potential for exposure via skin, subcutaneous, ingestion, or inhalation routes) must be considered for safe use of PDT by the healthcare professional. CONCLUSIONS: Engineering, administrative, and personal protective equipment controls are recommendations for the safe use and handling of PDT agents and light-emitting technologies.

Application of immunohistochemical staining to detect antigen destruction as a measure of tissue damage.

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, "antigen destruction immunohistochemistry" (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method.

Long-term adaptation of the human lung tumor cell line A549 to increasing concentrations of hydrogen peroxide.

Previously, we demonstrated that A549, a human lung cancer cell line, could be adapted to the free radical nitric oxide (NO●). NO● is known to be over expressed in human tumors. The original cell line, A549 (parent), and the newly adapted A549-HNO (which has a more aggressive phenotype) serve as a useful model system to study the biology of NO●. To see if tumor cells can similarly be adapted to any free radical with the same outcome, herein we successfully adapted A549 cells to high levels of hydrogen peroxide (HHP). A549-HHP, the resulting cell line, was more resistant and grew better then the parent cell line, and showed the following characteristics: (1) resistance to hydrogen peroxide, (2) resistance to NO●, (3) growth with and without hydrogen peroxide, and (4) resistance to doxorubicin. Gene chip analysis was used to determine the global gene expression changes between A549-parent and A549-HHP and revealed significant changes in the expression of over 1,700 genes. This gene profile was markedly different from that obtained from the A549-HNO cell line. The mitochondrial DNA content of the A549-HHP line determined by quantitative PCR favored a change for a more anaerobic metabolic profile. Our findings suggest that any free radical can induce resistance to other free radicals; this is especially important given that radiation therapy and many chemotherapeutic agents exert their effect via free radicals. Utilizing this model system to better understand the role of free radicals in tumor biology will help to develop new therapeutic approaches to treat lung cancer.

Part I. Development of a model system for studying nitric oxide in tumors: high nitric oxide-adapted head and neck squamous cell carcinoma cell lines.

The free radical nitric oxide (NO) is over-expressed in many tumors, including head and neck squamous cell carcinomas (HNSCC); however, the role NO plays in tumor pathophysiology is still not well understood. We, herein, report the development of an in vitro model system which can be used to probe the role of NO in the carcinogenesis of HNSCC. Five HNSCC cell lines were adapted to a high NO (HNO) environment by gradually introducing increasing concentrations of DETA-NONOate, a nitrogen-based NO donor, to cell media. The adaptation process was carried out until a sufficiently high enough donor concentration was reached which enabled the HNO cells to survive and grow, but which was lethal to the original, unadapted ("parent") cells. The adapted HNO cells exhibited analogous morphology to the parent cells, but grew better than their corresponding parent cells in normal media, on soft agar, and in the presence of hydrogen peroxide, an oxygen-based free radical donor. These results indicate that the HNO cell lines are unique and possess biologically different properties than the parent cell lines from which they originated. The HNO/parent cell lines developed herein may be used as a model system to better understand the role NO plays in HNSCC carcinogenesis.

Part II. Initial molecular and cellular characterization of high nitric oxide-adapted human tongue squamous cell carcinoma cell lines.

It is not understood why some head and neck squamous cell carcinomas, despite having identical morphology, demonstrate different tumor aggressiveness, including radioresistance. High levels of the free radical nitric oxide (NO) and increased expression of the NO-producing enzyme nitric oxide synthase (NOS) have been implicated in tumor progression. We previously adapted three human tongue cancer cell lines to high NO (HNO) levels by gradually exposing them to increasing concentrations of an NO donor; the HNO cells grew faster than their corresponding untreated ("parent") cells, despite being morphologically identical. Herein we initially characterize the HNO cells and compare the biological properties of the HNO and parent cells. HNO/parent cell line pairs were analyzed for cell cycle distribution, DNA damage, X-ray and ultraviolet radiation response, and expression of key cellular enzymes, including NOS, p53, glutathione S-transferase-pi (GST-pi), apurinic/apyrimidinic endonuclease-1 (APE1), and checkpoint kinases (Chk1, Chk2). While some of these properties were cell line-specific, the HNO cells typically exhibited properties associated with a more aggressive behavior profile than the parent cells (greater S-phase percentage, radioresistance, and elevated expression of GST-pi/APE1/Chk1/Chk2). To correlate these findings with conditions in primary tumors, we examined the NOS, GST-pi, and APE1 expression in human tongue squamous cell carcinomas. A majority of the clinical samples exhibited elevated expression levels of these enzymes. Together, the results herein suggest cancer cells exposed to HNO levels can develop resistance to free radicals by upregulating protective mechanisms, such as GST-pi and APE1. These upregulated defense mechanisms may contribute to their aggressive expression profile.

Synthesis and characterization of new porphyrazine-Gd(III) conjugates as multimodal MR contrast agents.

Magnetic resonance imaging (MRI) has long been used clinically and experimentally as a diagnostic tool to obtain three-dimensional, high-resolution images of deep tissues. These images are enhanced by the administration of contrast agents such as paramagnetic Gd(III) complexes. Herein, we describe the preparation of a series of multimodal imaging agents in which paramagnetic Gd(III) complexes are conjugated to a fluorescent tetrapyrrole, namely, a porphyrazine (pz). Zinc metalated pzs conjugated to one, four, or eight paramagnetic Gd(III) complexes are reported. Among these conjugates, Zn-Pz-8Gd(III) exhibits an ionic relaxivity four times that of the monomeric Gd(III) agent, presumably because of increased molecular weight and a molecular relaxivity that is approximately thirty times larger, while retaining the intense electronic absorption and emission of the unmodified pz. Unlike current clinical MR agents, Zn-Pz-1Gd(III) is taken up by cells. This probe demonstrates intracellular fluorescence by confocal microscopy and provides significant contrast enhancement in MR images, as well as marked phototoxicity in assays of cellular viability. These results suggest that pz agents possess a new potential for use in cancer imaging by both MRI and near-infrared (NIR) fluorescence, while acting as a platform for photodynamic therapy.

Nitric oxide: perspectives and emerging studies of a well known cytotoxin.

The free radical nitric oxide (NO(*)) is known to play a dual role in human physiology and pathophysiology. At low levels, NO(*) can protect cells; however, at higher levels, NO(*) is a known cytotoxin, having been implicated in tumor angiogenesis and progression. While the majority of research devoted to understanding the role of NO(*) in cancer has to date been tissue-specific, we herein review underlying commonalities of NO(*) which may well exist among tumors arising from a variety of different sites. We also discuss the role of NO(*) in human physiology and pathophysiology, including the very important relationship between NO(*) and the glutathione-transferases, a class of protective enzymes involved in cellular protection. The emerging role of NO(*) in three main areas of epigenetics-DNA methylation, microRNAs, and histone modifications-is then discussed. Finally, we describe the recent development of a model cell line system in which human tumor cell lines were adapted to high NO(*) (HNO) levels. We anticipate that these HNO cell lines will serve as a useful tool in the ongoing efforts to better understand the role of NO(*) in cancer.

Long-term adaptation of breast tumor cell lines to high concentrations of nitric oxide.

Nitric oxide (NO), a free radical, has been implicated in the biology of human cancers, including breast cancer, yet it is still unclear how NO affects tumor development and propagation. We herein gradually adapted four human breast adenocarcinoma cell lines (BT-20, Hs578T, T-47D, and MCF-7) to increasing concentrations of the NO donor DETA-NONOate up to 600 muM. The resulting model system consisted of a set of fully adapted high nitric oxide ("HNO") cell lines that are biologically different from the "parent" cell lines from which they originated. Although each of the four parent and HNO cell lines had identical morphologic appearance, the HNO cells grew faster than their corresponding parent cells and were resistant to both nitrogen- and oxygen-based free radicals. These cell lines serve as a novel tool to study the role of NO in breast cancer progression and potentially can be used to predict the therapeutic response leading to more efficient therapeutic regimens.

Chiral bis-acetal porphyrazines as near-infrared optical agents for detection and treatment of cancer.

We report the preparation of chiral oxygen atom-appended porphyrazines (pzs) as biomedical optical agents that absorb and emit in the near-IR wavelength range. These pzs take the form M[pz(A(4-n)B(n))], where "A" and "B" represent moieties appended to the pz's pyrrole entities, A = (2R,3R) 2,3-dimethyl-2,3-dimethoxy-1,4-diox-2-ene, B = beta,beta'-di-isopropoxybenzo, M is the incorporated metal ion (M = H(2), Zn), and n = 0, 1, 2 (-cis/-trans) and 3 (Scheme 1). When dissolved in polar media, H(2)[pz(trans-A(2)B(2))] 5a does not fluoresce and has a negligible quantum yield for singlet oxygen generation (capital EF, Cyrillic(Delta) = 0.074 +/- 0.001, methanol), as measured by the photo-oxidation of DMA. However, when sequestered in the nonpolar environment of a liposome, it displays strong NIR emission (lambda(max) = 705 nm, capital EF, Cyrillic(f) = 0.087) and an extremely high singlet oxygen quantum yield (capital EF, Cyrillic(Delta)-->1). Of this series, H(2)[pz(trans-A(2)B(2))] 5a is attractive as a potential optical probe, showing strongly fluorescent uptake by cells in culture, while 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide measurements of cell viability show no evidence of dark toxicity. This agent does show significant photoinduced toxicity suggesting that pzs such as 5a have promise as "theranostic" optical agents that can be visualized with fluorescence imaging while acting as a sensitizer for photodynamic therapy.

Characterization of a novel phantom for three-dimensional in vitro cell experiments.

A novel intensity-modulated radiation therapy (IMRT) phantom for use in three-dimensional in vitro cell experiments, based on a commercially available system (CIRS Inc., Norfolk, VA), was designed and fabricated. The water-equivalent plastic phantom can, with a set of water-equivalent plastic inserts, enclose 1-3 multi-well tissue culture plates. Dosimetry within the phantom was assessed using thermoluminescence dosimeters (TLDs) and film. The phantom was loaded with three tissue culture plates, and an array of TLDs or a set of three films was placed underneath each plate within the phantom, and then irradiated using an IMRT plan created for it. Measured doses from each dosimeter were compared to those acquired from the treatment planning system. The percent differences between TLD measurements and the corresponding points in the treatment plan ranged from 1.3% to 2.9%, differences which did not show statistical significance. Average point-by-point percent dose differences for each film plane ranged from 1.6% to 3.1%. The percentage dose difference for which 95% of the points in the film matched those corresponding to the calculated dose plane to within 3.0% ranged from 2.8% to 4.2%. The good agreement between predicted and measured dose shows that the phantom is a useful and efficient tool for three-dimensional in vitro cell experiments.

Synthesis and Biological Analysis of Thiotetra(ethylene glycol) monomethyl Ether-Functionalized Porphyrazines: Cellular Uptake and Toxicity Studies.

The porphyrazines (pzs), a class of porphyrin analogues, are being investigated for their potential use as tumor imaging/therapeutic agents. We here examine six peripherally-functionalized M[pz(AnB4-n)] pzs with n=4, 3, or 2 (in a trans conformation) and M = H2 or Zn, where A is an [S((CH2)2O)4Me]2 unit and B is a fused beta,beta'-diisopropyloxybenzo group. Cell viability/proliferation assays and fluorescence microscopy were carried out in both tumor and normal cells. Dark toxicity studies disclosed that four of the compounds exhibited toxicity in both normal and tumor cells; one was nontoxic in both normal and tumor cells, and one was selectively toxic to normal cells. Additionally, three of the pzs showed enhanced photo-induced toxicity with these effects in some cases being observed at treatment concentrations of up to ten-fold lower than that needed for a response in Photofrin. All six compounds were preferentially absorbed by tumor cells, suggesting that they have potential as in vitro diagnostic agents and as aids in the isolation and purification of aberrant cells from pathological specimens. In particular, two promising diagnostic candidates have been identified as part of this work.

Effect of proton pump inhibitor pantoprazole on growth and morphology of oral Lactobacillus strains.

OBJECTIVES: Proton pump inhibitors (PPI) used to suppress acid secretion in the stomach are among the most widely prescribed medications. There is emerging evidence of proton secretion elsewhere in the aerodigestive tract, and acidic microenvironments are integral to oral flora such as Lactobacillus. The hypothesis of this study is that the growth rate and morphology of oral Lactobacillus strains are effected by PPIs. METHODS: Nineteen different strains of Lactobacilli were inoculated in microtiter plates at pH of 4.5 to 6.5 and exposed to twofold dilutions of pantoprazole at a range of 2.5 mg/mL to 2.5 microg/mL. Bacterial growth was monitored, and the minimum inhibitory concentration (MIC) of the drug was determined for the strains most sensitive to pantoprazole. RESULTS: In the unexposed (control) group, nine Lactobacilli strains were affected by pH changes from 6.5 to 4.5. In the group exposed to pantoprazole, 9 of the 19 Lactobacilli strains were found to have an MIC below 625 microg/mL, with L. plantarum 14917 being the most sensitive (MIC = 20 microg/mL). In some strains, such as L. s. salivarius 11741, Gram-staining revealed conformational changes in the bacteria when grown in the presence of pantoprazole. CONCLUSION: Growth rates and morphology of oral Lactobacillus are affected by the pH of the environment. Pantoprazole at supraphysiologic doses further affects growth rates and conformation in some strains. SIGNIFICANCE: The balance of oral flora and upper digestive tract homeostasis may be affected by unexpected targets of PPI pharmacotherapy, with possible unanticipated consequences.

Gastroesophageal reflux disease (GERD): is there more to the story?

Gastroesophageal reflux disease (GERD) affects both men and women worldwide, with the most common symptom of GERD being frequent heartburn. If left untreated, more serious diseases including esophagitis and/or esophageal cancer may result. GERD has been commonly held to be the result of gastric acid refluxing into the esophagus. Recent work, however, has shown that there are acid-producing cells in the upper aerodigestive tract. In addition, acid-producing bacteria located within the upper gastrointestinal tract and oral cavity may also be a contributing factor in the onset of GERD. Proton pump inhibitors (PPIs) are commonly prescribed for treating GERD; these drugs are designed to stop the production of gastric acid by shutting down the H(+)/K(+)-ATPase enzyme located in parietal cells. PPI treatment is systemic and therefore significantly different than traditional antacids. Although a popular treatment choice, PPIs exhibit substantial interpatient variability and commonly fail to provide a complete cure to the disease. Recent studies have shown that H(+)/K(+)-ATPases are expressed in tissues outside the stomach, and the effects of PPIs in these nongastric tissues have not been fully explored. Likewise, acid-producing bacteria containing proton pumps are present in both the oral cavity and esophagus, and PPI use may also adversely affect these bacteria. The use of PPI therapy is further complicated by the two philosophical approaches to treating this disease: to treat only symptoms or to treat continuously. The latter approach frequently results in unwanted side effects which may be due to the PPIs acting on nongastric tissues or the microbes which colonize the upper aerodigestive tract.

Developing a structure-function relationship for anionic porphyrazines exhibiting selective anti-tumor activity.

The porphyrazines (pzs) are a class of porphyrin derivatives being studied for their use as optical imaging agents and photodynamic therapy (PDT) anti-tumor agents. A previous study revealed that the anionic pz, 18--of the form H2[pz(An;B4-n)], where A is [S(CH2)3CO2-], B is a fused beta',beta'-diisopropyloxy benzo group, with n=2 (trans)--selectively killed tumor cells, while analogous neutral and positively charged pzs lacked this property. In this report, we compare the properties of a suite of three H2[pz(An;B4-n)] pzs containing the same A and B groups as 18, but differing in their values of n: pzs 4 (n=4) and 11 (n=3), and 18 (n=2, trans) exhibit a progressive variation in charge due to the carboxylates, balance between hydrophobic/hydrophilic character, as well as a progressive variation in the singlet oxygen quantum yield (PhiDelta): PhiDelta (18)>PhiDelta (11)>PhiDelta (4). The biological activity of the pzs was tested in human lung carcinoma (A549) and SV40 transformed embryonic (WI-38 VA13) cell lines. Pzs 4 and 11 exhibited significant toxicity in both tumor and normal cells, while 18 showed selective anti-tumor cell activity in a dose-dependent manner. As the number of net negative charges decreased, the compounds became less toxic to normal cells, and the killing effect observed with these compounds was light independent. These observations indicate that the toxicity may have little to do with singlet oxygen quantum yields, but rather is more dependent on the net number of negative charges a pz contains. The study reported herein presents an example of how the porphyrazines can be easily modified to vary their biological behavior and specifically suggest that anionic porphyrazines pzs with lower n (fewer carboxylates, larger hydrophobic core) are more specific tumor killers, while those with larger n (increased net negative charge) are more potent tumor killers.

Charge dependence of cellular uptake and selective antitumor activity of porphyrazines.

Porphyrazines (pzs), or tetraazaporphyrins, can be viewed as porphyrinic macrocycles in which the porphyrin meso (CH) groups are replaced by nitrogen atoms; as such, it can be anticipated that pzs would show similar biocompatibility and biodistribution to those of porphyrins. However, distinctive chemical and physical features of the pzs differentiate them from either the porphyrins or phthalocyanines, in particular making them excellent candidates as optical imaging/therapeutic agents. The novelty of the pzs requires that we first determine how specific structures selectively alter biological function, leading to the development of "rules" that will be used to predict future biologically functional pzs. In the first of these studies, we present here a correlation of pz charge with biocompatibility for a suite of three pzs-neutral, negative, and positive. Confocal fluorescence microscopy and proliferation/viability measurements disclose that the three pzs differ in their toxicity, uptake, and localization in A549 human lung adenocarcinoma cells and WI-38 VA13 normal cells. Interestingly, the negatively charged pz exhibits selective dark toxicity in pulmonary adenocarcinoma cells.

ROM polymerization-capture-release strategy for the chromatography-free synthesis of novel unsymmetrical porphyrazines.

[reaction: see text] Crossover-Linstead macrocyclization reactions of two norbornenyl-tagged diaminomaleonitriles with dipropylmaleonitrile gave access to crude mixtures of porphyrazines containing diamino-hexapropyl-porphyrazine magnesium complexes. The mixtures were subjected to ring-opening metathesis polymerization to yield the insoluble diaminoporphyrazine-functionalized polymers. Acid-mediated cleavage from the polymer backbone followed by acylation of the resultant sensitive macrocyclic diamines gave monoacetyl-, monotrifluoroacetyl-, and ditrifluoroacetyl-substituted porphyrazinediamines. Conversion of these amido-porphyrazines to the corresponding zinc macrocycles and studies of their electronic absorption and emission spectra, electrochemistry, and photophysics are described.

Surface-bound porphyrazines: controlling reduction potentials of self-assembled monolayers through molecular proximity/orientation to a metal surface.

We report the preparation of two novel H2[pz(An;B(4-n))] porphyrazines (pzs) which were designed to position themselves quite differently when attached to a surface: one to form a standard self-assembled monolayer (SAM) roughly perpendicular to a surface, the other to lie horizontally along a surface. As the former, we synthesized a pz, 1, where one pyrrole group is functionalized with two thioethers terminated in mercaptides (SR, R = (CH2)3CONH(CH2)2S-), each protected as a disulfide, and -S-Me is attached to the other pyrrole sites; the latter is a pz, 2, with dialkoxybenzo groups fused to two trans-pyrroles of the pz ring, and SR groups are attached to the other pair of pyrroles. Nanostructures of 1 and 2 were successfully patterned on gold surfaces via dip-pen nanolithography, and the predicted molecular orientation of the resulting structures was confirmed by topographic AFM images. The two pzs exhibit similar reduction potentials in solution. Both show large shifts in potential upon surface binding, with the magnitude of the shift depending on the proximity/orientation of the pz to the surface. The first reduction potential of the "vertically" aligned 1 shifts by ca. +430 mV when incorporated in a binary pz/hexanethiol SAM, while that for 2, which lies flat, shifts by ca. +800 mV; the potential thus shifts by ca. +370 mV upon taking a given pz that stands atop a two-legged insulating "standoff" in a traditional SAM and "laying it down". We suggest these observed effects can be explained by image-charge energetics, and this is supported by a simple model.