Theranostic system for ratiometric fluorescence monitoring of peptide-guided targeted drug delivery

Conjugation of an anticancer drug with a cancer-specific carrier and a fluorescent dye to form a theranostic system enables real time monitoring of targeted drug delivery (TDD). However, the fluorescence signal from the dye is affected by the light absorption and scattering in the body, photobleaching, and instrumental parameters. Ratiometric measurements utilizing two fluorescence signals of different wavelengths are known to improve sensitivity, reliability and quantitation of fluorescence measurements in biological media. Herein, a novel theranostic system comprising the anticancer drug chlorambucil (CLB), cancer-specific peptide octreotide amide (OctA), and a long-wavelength dual fluorescent cyanine dye IRD enabling ratiometric monitoring of drug delivery was developed and evaluated on the cancer cell line PANC-1.

Novel theranostic system that first combines a cancer-targeting peptide with a long-wavelength dual fluorescent dye IRD in order to provide ratiometric monitoring of anticancer drug delivery is developed and evaluated in pancreatic cancer cell line.     

Protection from hypertension in mice by the Mediterranean diet is mediated by nitro fatty acid inhibition of soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) is inhibited by electrophilic lipids by their adduction to Cys521 proximal to its catalytic center. This inhibition prevents hydrolysis of the enzymes’ epoxyeicosatrienoic acid (EET) substrates, so they accumulate inducing vasodilation to lower blood pressure (BP). We generated a Cys521Ser sEH redox-dead knockin (KI) mouse model that was resistant to this mode of inhibition. The electrophilic lipid 10-nitro-oleic acid (NO₂-OA) inhibited hydrolase activity and also lowered BP in an angiotensin II-induced hypertension model in wild-type (WT) but not KI mice. Furthermore, EET/dihydroxy-epoxyeicosatrienoic acid isomer ratios were elevated in plasma from WT but not KI mice following NO₂-OA treatment, consistent with the redox-dead mutant being resistant to inhibition by lipid electrophiles. sEH was inhibited in WT mice fed linoleic acid and nitrite, key constituents of the Mediterranean diet that elevates electrophilic nitro fatty acid levels, whereas KIs were unaffected. These observations reveal that lipid electrophiles such as NO₂-OA mediate antihypertensive signaling actions by inhibiting sEH and suggest a mechanism accounting for protection from hypertension afforded by the Mediterranean diet.Soluble epoxide hydrolase (sEH) has a conserved cysteine (Cys521) proximal to its catalytic center. This cysteine can undergo Michael addition with electrophilic lipids, which inhibits hydrolysis of the enzyme’s epoxyeicosatrienoic acid (EET) substrates (1). This in turn elevates EET levels, which mediate blood vessel dilation and lowers blood pressure (BP), especially in the setting of hypertension (2, 3). Diverse sEH inhibitors limit injury in a variety of diseases (4), providing broad cardiovascular protection (5) against hypertension (6, 7), ischemia and reperfusion injury (8, 9), hypertrophy, and heart failure (10), as well as inflammation (11, 12). Consistent with the therapeutic potential of hydrolase inhibitors, sEH null mice are protected from pathological interventions (13). Conversely, genetic alterations that promote enhanced hydrolase activity are a risk factor for human heart failure (14).The endogenous lipid electrophile 10-nitrooctadec-9-enoic acid (nitro-oleic acid, NO₂-OA) inhibits sEH in vitro (1). NO₂-OA and other fatty acid nitroalkenes appear to signal via pleiotropic mechanisms including targeting and activating peroxisome proliferator-activated receptor gamma (PPARγ), the Kelch-like erythroid cell-derived protein with CNC homology (EHC)-associated protein-1 (Keap1), and nuclear factor (erythroid-derived)-like-2 (Nrf2)-regulated antioxidant response genes and inhibiting proinflammatory gene expression regulated by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (15, 16). Nitroalkenes are produced by radical addition of nitrogen dioxide (·NO₂) to one or more of the olefinic carbons of an unsaturated fatty acid. Nitrogen dioxide is both a product of oxidative inflammatory reactions involving nitric oxide (NO) and nitrite and the acidification nitrite. When the electron-withdrawing nitro group is bonded to alkenyl groups, this confers an electrophilic reactivity to fatty acids (17, 18). Thus, fatty acid nitroalkenes can modify proteins covalently via reversible Michael addition reactions that overall serves to link cellular metabolic and redox homeostasis with the posttranslational regulation of target protein function.Nitro fatty acids, which have been detected endogenously in plasma and urine of humans, animal models, and plants (19–21), mediate salutary cardiovascular signaling actions (22). For example they relax blood vessels, attenuate platelet activation, and reduce inflammation via cyclic guanosine monophosphate (cGMP)-independent mechanisms (23, 24). Of relevance, the Mediterranean diet is characterized by high consumption of unsaturated fatty acids, especially from olive oil and fish rich in oleic and linoleic acid, together with vegetables rich in nitrite and nitrate (25). The acidic and low-oxygen conditions in the stomach provide an environment for efficient nitration of such unsaturated fatty acids by nitrite (26).NO₂-OA normalizes blood pressure in an angiotensin (Ang) II-induced murine model of hypertension via undefined mechanisms (27). This was notable as pharmacological inhibitors of sEH also lower BP in murine hypertension, including salt- or Ang II-induced models (6, 7). As NO₂-OA inhibits sEH, we hypothesized that this mechanism may account for BP lowering in the setting of hypertension. Furthermore, as the Mediterranean diet both contains nitro fatty acids and can elevate their endogenous generation, this mechanism may contribute to dietary-induced BP decreases that in turn will reduce the risk of adverse cardiovascular event (28).Given the complexity of causally establishing whether nitro fatty acids lower BP by inhibiting sEH, especially in the setting of dietary-induced endogenous fatty acid nitration, we generated a Cys521Ser sEH knockin (KI) mouse. This “redox-inactive” sEH thiol mutant, rendered insensitive to adductive inhibition by lipid electrophiles in vitro, provided a novel model system for testing the impact of lipid nitroalkenes on sEH hydrolysis of vasoactive EET species and downstream physiological responses (1). The data reveal that nitro fatty acids, applied exogenously as a pharmacological agent or generated endogenously as part of the Mediterranean diet, inhibit sEH to elevate plasma EETs, which in turn lower BP.     

Sterilization analysis of contaminated healing abutments and impression copings

This study investigated sterilization of used implant impression copings and healing abutments. Components were analyzed after contamination with Enterococcus foecalis, followed by multiple rounds of sterilization by both steam autoclave and Chemiclave protocols. The authors' results demonstrated that used components showed sterility equal to new components without any visible distortion. These data suggest that component resterilization and reuse may be justified or at least considered in clinical practice. Also, implications for cost savings in the placement of implants are advanced.     

Effects of a late-life suicide risk--assessment training on multidisciplinary healthcare providers

Older adults are among the highest at risk for completing suicide, and they are more likely to seek mental health services from providers outside of traditional mental health care, but providers across the spectrum of care have limited training in suicide risk assessment and management and particularly lack training in suicide prevention for older adults. An educational program was developed to increase awareness and improve suicide risk assessment and management training for a range of healthcare providers who may see older adults in their care settings. One hundred thirty-two participants from two Veterans Affairs Medical Centers participated in a 6.5-hour-long workshop in the assessment and management of suicide risk in older adults. Participants were asked to complete pre- and postworkshop case notes and report on subjective changes in knowledge, attitudes, and confidence in assessment and managing suicide risk in older adults. Participants included social workers, nurses, physicians, psychologists, and occupational therapists from a variety of care settings, including outpatient and inpatient medical, outpatient and inpatient mental health, specialty clinics, home, and community. After the workshop, participants demonstrated improvement in the overall quality of case notes (P = .001), greater ability to recognize important conceptual suicide risk categories (P = .003), and reported heightened awareness of the importance of late-life suicide. The results suggest that educational training may have beneficial effect on the ability of multidisciplinary care providers to identify and manage suicide risk in elderly adults.     

DC-SIGN, C1q, and gC1qR form a trimolecular receptor complex on the surface of monocyte-derived immature dendritic cells

C1q modulates the differentiation and function of cells committed to the monocyte-derived dendritic cell (DC) lineage. Because the 2 C1q receptors found on the DC surface-gC1qR and cC1qR-lack a direct conduit into intracellular elements, we postulated that the receptors must form complexes with transmembrane partners. In the present study, we show that DC-SIGN, a C-type lectin expressed on DCs, binds directly to C1q, as assessed by ELISA, flow cytometry, and immunoprecipitation experiments. Surface plasmon resonance analysis revealed that the interaction was specific, and both intact C1q and the globular portion of C1q bound to DC-SIGN. Whereas IgG reduced this binding significantly, the Arg residues (162-163) of the C1q-A chain, which are thought to contribute to the C1q-IgG interaction, were not required for C1q binding to DC-SIGN. Binding was reduced significantly in the absence of Ca(2+) and by preincubation of DC-SIGN with mannan, suggesting that C1q binds to DC-SIGN at its principal Ca(2+)-binding pocket, which has increased affinity for mannose residues. Antigen-capture ELISA and immunofluorescence microscopy revealed that C1q and gC1qR associate with DC-SIGN on blood DC precursors and immature DCs. The results of the present study suggest that C1q/gC1qR may regulate DC differentiation and function through the DC-SIGN-mediated induction of cell-signaling pathways.     

The Difference in Self-Reported and Biological Measured HIV Prevalence: Implications for HIV Prevention

In Australia, HIV prevalence estimates among gay men have been mainly based on self-reported HIV status collected in annual behavioural surveys. We measured biological HIV prevalence among gay men in Melbourne, Australia, using a facility based sampling method. We calculated HIV prevalence and used logistic regression to assess correlates of a positive HIV test. A total of 639 gay men were recruited completed a survey and provided oral fluid for HIV testing from seven venues in 2008. The median age of the participants was 35 years (range 18-75 years). Overall biological HIV prevalence was 9.5% (95% CI 7.5-12.0%) compared with 6.3% (95% CI 4.5-8.4%) for self-reported HIV positive status. We found a significant discrepancy between test detected biological and self-report HIV status in our study, with 19 men (31.1%) unaware of their HIV infection. These results highlight the importance of repeatable biological estimates to inform and evaluate HIV prevention strategies.     

Transgastric large-organ extraction: the initial human experience

Introduction

In laparoscopy, it often is the case that port sites are enlarged for specimen extraction. This leads to higher risk of trocar site complications, such as infection or incisional hernia. Natural orifice surgery (NOTES) is beneficial for minimizing these complications, and this is emphasized when the extracted specimen is of large volume. We have been using transgastric technique for appendectomy, cholecystectomy, and laparoscopic sleeve gastrectomy (LSG). Of these transgastric operations, we focus on the one with relatively large-organ extraction: LSG with transoral remnant extraction (TORE). We describe the details and feasibility of this procedure and compare the outcomes to conventional LSG.

Methods

All patients undergoing LSG were considered candidates for TORE and were consented for this procedure if interested after an informed discussion. Eighteen LSGs with TORE (TORE group) and ten conventional LSGs (non-TORE group) were performed from August 2010 to March 2011. We retrospectively compared these two groups for the age, sex, preoperative body mass index, operating room time, hospital stay, excess weight loss (EWL), and trocar site complications. Laparoscopic sleeve gastrectomy with TORE consists of conventional LSG and transgastric retrieval of the resected stomach. The procedure exceeds exactly the same manner as conventional LSG until the initial stapling of the stomach. For TORE, the gastrectomy is initiated 5 cm proximal to the pylorus than usual LSG to save the space for the gastrotomy used for specimen retrieval. After the gastrectomy is completed, the full thickness of the distal most part of the staple line is incised open as wide as 2 cm by using electric cautery or ultrasonic dissector. A flexible upper endoscope, which has been in the stomach already as a bougie for gastrectomy, is then guided into the peritoneal cavity through the gastrotomy. The specimen is grasped endoscopically with a snare and extracted transorally. Following this, the gastrotomy is closed laparoscopically. The final shape of the gastric sleeve is identical to the one of conventional LSG.

Results

There was no significant difference between the TORE and the non-TORE group for patients’ profile, operating room time, hospital stay, and EWL. Neither group has experienced perioperative complications. All specimens were extracted readily and safely in the TORE group. Of the ten cases in the non-TORE group, four required extension of the trocar site. No trocar site complications were found in the TORE group, whereas the extended trocar site developed panniculitis in two cases of the non-TORE group; one required panniculectomy for refractory induration.

Conclusions

TORE can be safely and easily performed by surgeons with laparoscopic and endoscopic skill, and with commonly available instruments. While producing identical outcomes, our initial experience with the TORE technique demonstrates an advantage over traditional LSG, because it minimizes trocar site complications. Transgastric organ extraction is potentially applicable to other large-organ extractions in laparoscopic surgery without excessive risk or resources. Larger case volume and longer follow-up period is awaited.