Search

The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane proteins. Here, we leveraged recent advances in cryo-electron microscopy (cryo-EM) and structure prediction to derive insights into several novel configurations of the ribosome-translocon complex. We show how a transmembrane domain (TMD) in a looped configuration passes through the Sec61 lateral gate during membrane insertion; how a nascent chain can bind and constrain the conformation of ribosomal protein uL22; and how the translocon-associated protein (TRAP) complex can adjust its position during different stages of protein biogenesis. Most unexpectedly, we find that a large proportion of translocon complexes contains RAMP4 intercalated into Sec61's lateral gate, widening Sec61's central pore and contributing to its hydrophilic interior. These structures lead to mechanistic hypotheses for translocon function and highlight a remarkably plastic machinery whose conformations and composition adjust dynamically to its diverse range of substrates., Competing Interests: AL, FZ, RK No competing interests declared, RH Scientific advisor and equity holder of Gate Bioscience, (© 2024, Lewis et al.)

Nanoparticle carriers enable the multivalent delivery of nucleic acids to cells and protect them from degradation. In this chapter, we present a comprehensive overview of four methodologies: electrophoretic mobility shift assay (EMSA), alamarBlue/CFDA-AM cell viability dyes, fluorescence microscopy, and antiviral assays, which collectively are tools to explore interactions between nucleic acids and nanoparticles, and their biological efficacy. These assays provide insights into binding potential, cytotoxicity, and antiviral efficacy of nucleic acid-based nanoparticle treatments furthering the development of effective antiviral therapeutics., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Laparoscopic and robotic-assisted approaches to hepatopancreatobiliary (HPB) operations have expanded worldwide. As surgeons and medical centers contemplate initiating and expanding minimally invasive surgical (MIS) programs for complex HPB surgical operations, there are many factors to consider. This review highlights the key components of developing an MIS HPB program and shares our recent institutional experience with the adoption and expansion of an MIS approach to pancreaticoduodenectomy., (© 2023 The Authors. Journal of Surgical Oncology published by Wiley Periodicals LLC.)

Culicoides biting midges (Diptera: Ceratopogonidae) are hematophagous flies that transmit several viruses of veterinary concern to livestock. Understanding blood feeding behaviors is integral towards identification of putative vector species and preventing the transmission of these pathogens. PCR-based blood meal analysis was conducted on 440 blood-engorged Culicoides midges collected in northeastern Kansas, with 316 (71.8%) returning non-human vertebrate identifications at the ≥95% identity match level. Broadly, Culicoides sonorensis , Culicoides stellifer , and Culicoides variipennis were found to feed heavily on mammalian hosts, while Culicoides crepuscularis and Culicoides haematopotus fed on avian hosts. The blood meals in all specimens were graded prior to DNA extraction to determine whether blood meal size or digestion status significantly impacted the likelihood of a quality host match. Size had a significant impact on the likelihood of a quality match at grades 3-5, whereas digestion only significantly impacted outcomes at the most extreme grade. These vector-host dynamics have not previously been studied in Culicoides collected in Kansas, which represents a unique tallgrass prairie biome within the United States that is heavily interspersed with livestock operations. Based on these data, the highly abundant species C. crepuscularis and C. haematopotus are unlikely to be major vectors of mammalian viruses.

Surgery is the cornerstone of treatment for retroperitoneal sarcoma (RPS). Surgery should be performed by a surgical oncologist with sub-specialization in this disease and in the context of a multidisciplinary team of sarcoma specialists. For primary RPS, the goal of surgery is to achieve the complete en bloc resection of the tumor along with involved organs and structures to maximize the clearance of the disease. The extent of resection also needs to consider the risk of complications. Unfortunately, the overarching challenge in primary RPS treatment is that even with optimal surgery, tumor recurrence occurs frequently. The pattern of recurrence after surgery (e.g., local versus distant) is strongly associated with the specific histologic type of RPS. Radiation and systemic therapy may improve outcomes in RPS and there is emerging data studying the benefit of non-surgical treatments in primary disease. Topics in need of further investigation include criteria for unresectability and management of locally recurrent disease. Moving forward, global collaboration among RPS specialists will be key for continuing to advance our understanding of this disease and find more effective treatments.

Multipass membrane proteins play numerous roles in biology and include receptors, transporters, ion channels and enzymes 1,2 . How multipass proteins are co-translationally inserted and folded at the endoplasmic reticulum is not well understood 2 . The prevailing model posits that each transmembrane domain (TMD) of a multipass protein successively passes into the lipid bilayer through a front-side lateral gate of the Sec61 protein translocation channel 3-9 . The PAT complex, an intramembrane chaperone comprising Asterix and CCDC47, engages early TMDs of multipass proteins to promote their biogenesis by an unknown mechanism 10 . Here, biochemical and structural analysis of intermediates during multipass protein biogenesis showed that the nascent chain is not engaged with Sec61, which is occluded and latched closed by CCDC47. Instead, Asterix binds to and redirects the substrate to a location behind Sec61, where the PAT complex contributes to a multipass translocon surrounding a semi-enclosed, lipid-filled cavity 11 . Detection of multiple TMDs in this cavity after their emergence from the ribosome suggests that multipass proteins insert and fold behind Sec61. Accordingly, biogenesis of several multipass proteins was unimpeded by inhibitors of the Sec61 lateral gate. These findings elucidate the mechanism of an intramembrane chaperone and suggest a new framework for multipass membrane protein biogenesis at the endoplasmic reticulum., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Protein transporters translocate hydrophilic segments of polypeptide across hydrophobic cell membranes. Two protein transporters are ubiquitous and date back to the last universal common ancestor: SecY and YidC. SecY consists of two pseudosymmetric halves, which together form a membrane-spanning protein-conducting channel. YidC is an asymmetric molecule with a protein-conducting hydrophilic groove that partially spans the membrane. Although both transporters mediate insertion of membrane proteins with short translocated domains, only SecY transports secretory proteins and membrane proteins with long translocated domains. The evolutionary origins of these ancient and essential transporters are not known., Results: The features conserved by the two halves of SecY indicate that their common ancestor was an antiparallel homodimeric channel. Structural searches with SecY's halves detect exceptional similarity with YidC homologs. The SecY halves and YidC share a fold comprising a three-helix bundle interrupted by a helical hairpin. In YidC, this hairpin is cytoplasmic and facilitates substrate delivery, whereas in SecY, it is transmembrane and forms the substrate-binding lateral gate helices. In both transporters, the three-helix bundle forms a protein-conducting hydrophilic groove delimited by a conserved hydrophobic residue. Based on these similarities, we propose that SecY originated as a YidC homolog which formed a channel by juxtaposing two hydrophilic grooves in an antiparallel homodimer. We find that archaeal YidC and its eukaryotic descendants use this same dimerisation interface to heterodimerise with a conserved partner. YidC's sufficiency for the function of simple cells is suggested by the results of reductive evolution in mitochondria and plastids, which tend to retain SecY only if they require translocation of large hydrophilic domains., Conclusions: SecY and YidC share previously unrecognised similarities in sequence, structure, mechanism, and function. Our delineation of a detailed correspondence between these two essential and ancient transporters enables a deeper mechanistic understanding of how each functions. Furthermore, key differences between them help explain how SecY performs its distinctive function in the recognition and translocation of secretory proteins. The unified theory presented here explains the evolution of these features, and thus reconstructs a key step in the origin of cells., (© 2021. The Author(s).)

Objective: The main goal of this investigation is to compare the Greater Occipital Nerve (GON) and Supra Orbital Nerve (SON) block methods used in the treatment of migraine attacks in the ED with each other, in combination, and with a placebo., Methods: This study was planned as a single center, prospective, double-blind, randomized control study. The patients were divided into 4 groups: GON, SON, Combined, and Placebo. Groups were named according to the nerve areas that were blocked. Therefore1% lidocaine for nerve blockade and 0.9% NaCl for placebo effect was used. Along with the time of admission, baseline pain scale values, as well as recordings at 30 and 60 min in addition to the Visual Analog Scale (VAS) at the 120th min. and Likert-Type (LT) Verbal scale at the 120th min. were measured. The primary outcome of this study was a change in the VAS and LT values at the 0-120th minute after treatment RESULTS: In this study, 128 patients in 4 groups were included in the analysis. In the GON group, SON group, and Combined group, the change observed at the 120th minute scores compared to baseline VAS scores was higher than Placebo group [OR (95% CI) = -17.4 (-24.8, -9.9), 32.1 (23.8, 40.3), 49.5 (41.9, 57), respectively]. In inter-group comparison, it was found that the VAS and LT scores of the Combined group and the GON group improved at a higher rate than the SON group CONCLUSIONS: SON blockade, GON blockade, and a combination of these two blockades are effective treatment methods in acute migraine attack. Performing a GON or Combined blockade for migraine attack causes greater pain reduction than SON blockade., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: The aim of this study was to determine outcomes of primary tumor resection in metastatic neuroendocrine tumors across all primary tumor sites., Background: Primary tumor resection (PTR) may offer a survival benefit in metastatic gastrointestinal neuroendocrine tumors (GI-NETs); however, few studies have examined the effect of primary site and grade on resection and survival., Methods: This is a retrospective study of patients with metastatic GI-NETs at presentation between 2005 and 2011 using the California Cancer Registry (CCR) dataset merged with California Office of Statewide Health Planning and Development (OSHPD) inpatient longitudinal database. Primary outcome was overall survival (OS). Univariate and multivariate (MV) analyses were performed using the Pearson Chi-squared tests and Cox proportional hazard, respectively. OS was estimated using the Kaplan-Meier method and log-rank test., Results: A total of 854 patients with GI-NET metastases on presentation underwent 392 PTRs. Liver metastases occurred in 430 patients; 240 received liver treatment(s). PTR improved OS in patients with untreated metastases (median survival 10 vs 38 months, P < 0.001). On MV analysis adjusted for demographics, tumor stage, grade, chemotherapy use, Charlson comorbidity index, primary tumor location, or treatment of liver metastases, PTR with/without liver treatment improved OS in comparison to no treatment [hazard ratio (HR) 0.50, P < 0.001 and 0.39, P < 0.001, respectively]. PTR offered a survival benefit across all grades (low-grade, HR 0.38, P = 0.002 and high-grade, HR 0.62, P = 0.025) CONCLUSION:: PTR in GI-NET is associated with a better survival, with or without liver treatment, irrespective of grade. This study supports the resection of the primary tumor in patients with metastatic GI-NETs, independent of liver treatment.

The microbiome is defined as the collective genomes of the microbes (composed of bacteria, bacteriophage, fungi, protozoa, and viruses) that colonize the human body, and alterations have been associated with a number of disease states. Changes in gut commensals can influence the neurologic system via the brain-gut axis, and systemic insults such as trauma or traumatic brain injury (TBI) may alter the gut microbiome. The objective of this study was to evaluate the gut microbiome in a preclinical TBI cortical impact model. Male rats underwent craniotomy and randomized to a sham group (n = 4), or a moderate TBI (n = 10) using a pneumatic impactor. MRI and behavioral assessments were performed pre-TBI and on days 2, 7, and 14 days thereafter. Microbiome composition was determined with 16s rRNA sequencing from fecal sample DNA pre-TBI and 2 hrs, 1, 3, and 7 days afterward. Alpha- and β-bacterial diversity, as well as organizational taxonomic units (OTUs), were determined. Significant changes in the gut microbiome were evident as early as 2 h after TBI as compared with pre-injured samples and sham rats. While there were varying trends among the phylogenetic families across time, some changes persisted through 7 days in the absence of therapeutic intervention. While large structural lesions and behavioral deficits were apparent post-TBI, there were modest but significant decreases in α-diversity. Moreover, both changes in representative phyla and α-diversity measures were significantly correlated with MRI-determined lesion volume. These results suggest that changes in the microbiome may represent a novel biomarker to stage TBI severity and predict functional outcome.

Background: This study characterizes the gastrointestinal (GI) microbiome in a pre-clinical polytrauma hemorrhage model., Methods: Rats (n = 6) were anesthetized, hemorrhaged 20% of their blood volume, and subjected to a femur fracture and crush injuries to the small intestine, liver, and limb skeletal muscle without resuscitation. Fecal samples were collected pre-injury and 2 h post-injury. Purified DNA from the samples underwent 16s rRNA sequencing for microbial quantification. Bacterial diversity analysis and taxonomic classification were performed., Results: Following injury, the gut microbial composition was altered with a shift in beta diversity and significant differences in the relative abundance of taxa. The relative abundance of the families Lachnospiraceae and Mogibacteriaceae was increased at 2 h, while Barnesiellaceae and Bacteroidaceae were decreased. Alpha diversity was unchanged., Conclusions: The GI microbiome is altered in rats subjected to a polytrauma hemorrhage model at 2 h post-injury in the absence of antibiotics or therapeutic interventions., (Copyright © 2018. Published by Elsevier Inc.)

The field of printed electronics is continually trying to reduce the dimensions of the electrical components. Here, a method of printing metallic lines with widths as small as 15 nm and up to a few micrometers using fountain pen nanolithography (FPN) is shown. The FPN technique is based on a bent nanopipette with atomic force feedback that acts similar to a nanopen. The geometry of the nanopen allows for rapid placement accuracy of the printing tip, on any desired location, with the highest of optical sub-micrometer resolution. Using this nanopen, investigations of various inks are undertaken together with instrumental and script-tool development that allows accurate printing of multiple layers. This has led to the printing of conductive lines using inks composed of silver nanoparticles and salt solutions of silver and copper. In addition, it is shown that the method can be applied to substrates of various materials with minimal effect on the dimension of the line. The line widths are varied by using nanopens with different orifices or by tailoring the wetting properties of the ink on the substrate. Metallic interconnections of conducting lines are reported., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: The benefits of minimally invasive distal pancreatectomy (MIDP) over open surgery continue to be investigated. Frailty is a known predictor of postoperative outcome. We hypothesized that the benefit of minimally invasive distal pancreatectomy is the greatest for the frailest of patients., Methods: Data from the pancreas-targeted National Surgical Quality Improvement Program (NSQIP) database for 2014 were reviewed. A modified frailty index (mFI) with 11 preoperative variables previously validated for use in NSQIP was used to determine the correlation between frailty and postoperative outcomes, including Clavien grade IV complications. Patients were classified into non-frail (mFI = 0) or frail (mIF > 0), in which they were subclassified into mildly frail (mFI 1 or 2) or severely frail (mFI = 3)., Results: A total of 1,038 distal pancreatectomies (DP) were included in the analysis, of which 387 were minimally invasive (MIDP: laparoscopic: 285, robotic: 102), 558 open DP (ODP), and 93 MIDP converted to open (MIDPcODP: laparoscopic: 80, robotic: 13). More than 90% of patients had an mFI of 0 or 1 (mFI 0 = 473 (45.6%), 1 = 466 (44.9%), 2 = 94 (9.1%), and 3 = 5 (0.5%), respectively). Overall, 4.6% of patients experienced Clavien grade IV complications and 1.1% a mortality. Non-frail patients experienced a similar rate of grade IV Clavien complications with MIDP vs. ODP vs. MIDPcOP (2.3 vs. 2.3 vs. 4.9%; p = 0.6), whereas frail patients (mFI > 0) had a lower rate of complications with MIDP (2.4 vs. 8.3 vs. 11.5; p = 0.007). Worsening frailty correlated with an increase in complications (non-frail: 2.5%; mildly frail: 6.3%; severely frail: 20%; p = 0.005)., Conclusion: MIDP is associated with a lower risk of Clavien grade IV complications compared to ODP for frail patients, especially for benign disease. Thus, minimally invasive approach may mitigate risk in frail patients.

The incidence of pancreatic neuroendocrine tumors (PNETs) has significantly increased in recent years, although this may reflect better detection and improved diagnosis, in addition to a true increase in incidence. The majority of PNETs are nonfunctional; up to half of nonmetastatic PNETs will present incidentally, and 85% will develop metastases over their lifetime. Treatment of PNETs is largely dictated by their heterogeneous nature and usually indolent behavior. Surgery is a mainstay of treatment, both in early PNETs and in metastatic disease. In this review, we focus on the treatment of well-differentiated early and metastatic PNETs, emphasizing current controversies, recent advances in therapy, and the multidisciplinary approach required for optimal treatment.

The present study aims to evaluate the role of laparoscopy in frail patients undergoing colorectal surgery for colorectal cancer. A review of the 2011 to 2014 American College of Surgeons National Surgical Quality Improvement Program database was performed to identify frail patients (using a frailty index), who underwent resection for colorectal cancer. Univariable and multivariable analyses were performed to evaluate 30-day mortality and Clavien-Dindo grade IV (CD-IV) complications. A total of 52,087 patients with colorectal cancer were identified, of which frailty accounted for 2.63 per cent (index score ≥5). Patients above the age 85 were considered frail 6.8 per cent of the time and accounted for 24.5 per cent of patients with frailty. Laparoscopic surgery was performed in 32.9 and 53.1 per cent of patients with and without frailty (P < 0.001). Patients with frailty were less likely to die within 30 days of surgery if younger (P = 0.004), performed electively (P < 0.001), or laparoscopically (P < 0.001). On multivariate analysis, laparoscopy and elective surgery were associated with better perioperative survival; whereas, older age, male sex, and tobacco use were associated with 30-day mortality. Laparoscopy and lower body mass index were associated with fewer Clavien-Dindo grade IV complications. Although laparoscopy is performed less commonly in the frail, this study indicated better perioperative outcomes for patients undergoing elective surgery who were <85 years old.

The ability to control the energy flow of light at the nanoscale is fundamental to modern communication and big-data technologies, as well as quantum information processing schemes. However, since photons are diffraction-limited, efforts of confining them to dimensions of integrated electronics have so far proven elusive. A promising way to facilitate nanoscale manipulation of light is through plasmon polaritons-coupled excitations of photons and charge carriers. These tightly confined hybrid waves can facilitate compression of optical functionalities to the nanoscale but suffer from huge propagation losses that limit their use to mostly subwavelength scale applications. With only weak evidence of macroscale plasmon polaritons, propagation has recently been reported theoretically and indirectly, no experiments so far have directly resolved long-range propagating optical plasmons in real space. Here, we launch and detect nanoscale optical signals, for record distances in a wireless link based on novel plasmonic nanotransceivers. We use a combination of scanning probe microscopies to provide high resolution real space images of the optical near fields and investigate their long-range propagation principles. We design our nanotransceivers based on a high-performance nanoantenna, Plantenna, hybridized with channel plasmon waveguides with a cross-section of 20 nm × 20 nm, and observe propagation for distances up to 1000 times greater than the plasmon wavelength. We experimentally show that our approach hugely outperforms both waveguide and wireless nanophotonic links. This successful alliance between Plantenna and plasmon waveguides paves the way for new generations of optical interconnects and expedites long-range interaction between quantum emitters and photomolecular devices.

Force controlled optical imaging of membranes of living cells is demonstrated. Such imaging has been extended to image membrane potential changes to demonstrate that live cell imaging has been achieved. To accomplish this advance, limitations inherent in atomic force microscopy (AFM) since its inception in 1986 [G. Binnig, C. F. Quate, and C. Gerber, "Atomic Force Microscope," Phys. Rev. Lett. 56, 930-933 (1986).] had to be overcome. The advances allow for live cell imaging of a whole genre of functional biological imaging with stiff (1-10N/m) scanned probe imaging cantilevers. Even topographic imaging of fine cell protrusions, such as microvilli, has been accomplished with such cantilevers. Similar topographic imaging has only recently been demonstrated with the standard soft (0.05N/m) cantilevers that are generally required for live cell imaging. The progress reported here demonstrates both ultrasensitive AFM (~100pN), capable of topographic imaging of even microvilli protruding from cell membranes and new functional applications that should have a significant impact on optical and other approaches in biological imaging of living systems and ultrasoft materials.

Background: In order to decrease cost and increase healthcare quality there is an ongoing effort to reduce readmissions after complex operations. The timing and severity of post-discharge complications after hepatectomy need to be better defined., Methods: All patients undergoing liver resection at a single institution from Jan 2009-Jun 2015 were included. Complications were scored using the comprehensive complication index (CCI). Multivariate analysis is performed using logistic regression. Receiver operating characteristics (ROCs) were analyzed to optimize the Readmission Risk Index (RRI)., Results: Of the 258 patients that met the inclusion criteria, 26 (10%) were readmitted within 30 days of discharge after hepatectomy. On multivariate analysis age ≥68 years (OR 3.76; 95% CI 1.47-9.63, p = 0.006), CCI ≥ 15 (OR 3.65; 95% CI 1.39-9.56, p = 0.008), maximum temperature within 48 h of discharge (OR 3.02; 95% CI 1.17-7.75, p = 0.022) and white blood cell count ≥10.2 billion cells/L within 48 h of discharge (OR 2.88; 95% CI 1.04-8, p = 0.043) were independent predictors of 30-day readmission. These variables were used to develop the RRI (ROC area 0.80; 95% CI 0.70-0.9, p < 0.001)., Conclusion: The CCI and pre-discharge variables can help identify individuals at risk of readmission. Readmission risk reduction efforts should focus on this subset of patients., (Copyright © 2017 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Mass casualty incidents (MCIs) are events resulting in more injured patients than hospital systems can handle with standard protocols. Several studies have assessed hospital preparedness during MCIs. However, physicians and trauma surgeons need to be familiar with their hospital's MCI Plan. The purpose of this survey was to assess hospitals' and trauma surgeon's preparedness for MCIs. Online surveys were e-mailed to members of the American College of Surgeons committee on Trauma Ad Hoc Committee on Disaster and Mass Casualty Management before the March 2012 meeting. Eighty surveys were analyzed (of 258). About 76 per cent were American College of Surgeons Level I trauma centers, 18 per cent were Level II trauma centers. Fifty-seven per cent of Level I and 21 per cent of Level II trauma centers had experienced an MCI. A total of 98 per cent of respondents thought it was likely their hospital would see a future MCI. Severe weather storm was the most likely event (95%), followed by public transportation incident (86%), then explosion (85%). About 83 per cent of hospitals had mechanisms to request additional physician/surgeons, and 80 per cent reported plans for operative triage. The majority of trauma surgeons felt prepared for an MCI and believed an event was likely to occur in the future. The survey was limited by the highly select group of respondents and future surveys will be necessary.

Background: Gamma delta T-cells have been shown to be important to the early immunoinflammatory response to injury, independent of infection. This unique T-cell population acts to regulate cell trafficking and the release of cytokines and growth factors. We propose this sterile inflammatory response is in part associated with damage associated molecular patterns (DAMPs) generated by major injury, such as burn, and mediated via toll-like receptors (TLRs). It is unknown whether DAMPs can activate resident γδ T-cells that reside in skin., Methods: Gamma delta T-cells were isolated from the skin of male C57BL/6 mice by enzymatic digestion. Mitochondrial DAMPs (MTDs) were generated from mitochondria isolated from mouse livers by sonication and centrifugation. Dermal γδ T-cells were incubated with MTDs (0-500 μg/ml) for 24 hr and cells and supernatants were collected for analysis., Results: MTDs activated dermal γδ T-cells, as evidenced by increased TLR2 and TLR4 expression following in vitro exposure. MTDs also induced the production of inflammatory cytokines (IL-1β, IL-6), and growth factors (PDGF and VEGF) by γδ T-cells., Conclusions: These findings herein support the concept that MTDs released after tissue/cellular injury are capable of activating dermal γδ T-cells. We propose that the activation of this unique T-cell population is central in the initiation of sterile inflammation and also contributes to the subsequent healing processes.

Genome engineering technologies now enable precise manipulation of organism genotype, but can be limited in scalability by their design requirements. Here we describe Merlin ( http://merlincad.org ), an open-source web-based tool to assist biologists in designing experiments using multiplex automated genome engineering (MAGE). Merlin provides methods to generate pools of single-stranded DNA oligonucleotides (oligos) for MAGE experiments by performing free energy calculation and BLAST scoring on a sliding window spanning the targeted site. These oligos are designed not only to improve recombination efficiency, but also to minimize off-target interactions. The application further assists experiment planning by reporting predicted allelic replacement rates after multiple MAGE cycles, and enables rapid result validation by generating primer sequences for multiplexed allele-specific colony PCR. Here we describe the Merlin oligo and primer design procedures and validate their functionality compared to OptMAGE by eliminating seven AvrII restriction sites from the Escherichia coli genome.

Plasmonic gold nanoparticles (AuNPs) are widely investigated for cancer therapy, due to their ability to strongly absorb light and convert it to heat and thus selectively destroy tumor cells. In this study we shed light on a new aspect of AuNPs and their plasmonic excitation, wherein they can provide anti-oxidant and anti-inflammatory protection by stimulating the cellular protective Nrf2 pathway. Our study was carried out on cells of the immune system, macrophages, and on skin cells, keratinocytes. A different response to AuNPs was noted in the two types of cells, explained by their distinct uptake profiles. In keratinocytes, the exposure to AuNPs, even at low concentrations, was sufficient to activate the Nrf2 pathway, without any irradiation, due to the presence of free AuNPs inside the cytosol. In contrast, in macrophages, the plasmonic excitation of the AuNPs by a low, non-lethal irradiation dose was required for their release from the constraining vesicles. The mechanism by which AuNPs activate the Nrf2 pathway was studied. Direct and indirect activation were suggested, based on the inherent ability of the AuNPs to react with thiol groups and to generate reactive oxygen species, in particular, under plasmonic excitation. The ability of AuNPs to directly activate the Nrf2 pathway renders them good candidates for treatment of disorders in which the up-regulation of Nrf2 is beneficial, specifically for topical treatment of inflammatory skin diseases.

Malignant parotid tumors are heterogeneous and diverse. Accurate diagnosis requires a pathologist familiar with the various histologic subtypes, immunohistochemistry stains, and common translocations. Clinical course varies according to tumor subtype, ranging from indolent, slow-growing adenoid cystic carcinoma to rapidly progressive, possibly fatal, salivary ductal carcinoma. Histologic grade is important in prognosis and therapy. Surgery remains the mainstay of treatment when negative margins can be achieved. Radiation improves locoregional control of tumors with high-risk features. Chemotherapy for parotid tumors can be disappointing. Studies of new targeted therapies have not offered significant benefits., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Single-walled carbon nanotubes (SWCNTs) are considered pivotal components for molecular electronics. Techniques for SWCNT lithography today lack simplicity, flexibility, and speed of direct, oriented deposition at specific target locations. In this paper SWCNTs are directly drawn and placed with chemical identification and demonstrated orientation using fountain pen nanolithography (FPN) under ambient conditions. Placement across specific electrical contacts with such alignment is demonstrated and characterized. The fundamental basis of the drawing process with alignment has potential applications for other related systems such as inorganic nanotubes, polymers, and biological molecules.

Purpose: To evaluate the impact of previous liver resection on the safety of resin microsphere radioembolization (RE)., Materials and Methods: A single-center retrospective review was performed of 22 patients who underwent resin microsphere RE after liver resection during the period 2009-2014. Prescribed patient dose using the body surface area (BSA) model and a theoretical dose calculated from the actual liver volume on imaging were recorded. Patient and treatment characteristics were analyzed for factors that contributed to toxicity., Results: In 13 patients, 20 grade 1-3 toxicities were identified. No differences in toxicity were seen based on extent of prior hepatic resection or whether whole-liver treatments were performed (P = .2). The measured liver volume based on cross-sectional imaging correlated poorly with the expected liver volume based on BSA (r = 0.43). After adjusting for the patients' measured liver volume on cross-sectional imaging, five patients were determined to be relatively overdosed and seven patients were determined to be relatively underdosed by the BSA method. Despite these differences, no association was found with patient toxicities and either an overestimation or an underestimation of liver volume (P = .4)., Conclusion: Previous hepatic resection does not adversely alter the safety profile of yttrium-90 RE. BSA poorly predicts expected liver volume in this population. However, standard BSA-based dosing and whole-liver remnant treatments do not increase hepatotoxicity., (Copyright © 2016 SIR. Published by Elsevier Inc. All rights reserved.)

There has been a significant increase in the incidence of human papillomavirus (HPV)-mediated oropharyngeal cancer in the United States. This entity is most commonly diagnosed in nonsmoking middle-aged white males. The majority of the patients present with asymptomatic, persistent neck masses despite antibiotic therapy. An awareness of this condition and a high degree of suspicion is necessary for timely diagnosis. HPV-mediated oropharyngeal squamous cell carcinomas (HPV-OPSCCs) are unique biologically and clinically, and affected patients enjoy better outcomes with existing standard therapies than do patients with OPSCC mediated by tobacco exposure. The p16 protein is usually overexpressed in HPV-OPSCC, and its detection on immunohistochemistry is a reliable surrogate marker for this disease. In this review, we discuss current paradigms in the diagnosis and management of HPV-OPSCC, and we emphasize pertinent research questions to investigate going forward, including whether to deintensify treatment in these patients.

Background: Activation of the SAPK/MAPK signaling pathway by pro-inflammatory cytokines is known to induce apoptosis in cultured articular chondrocytes. C-28/I2, an immortalized human juvenile chondrocyte cell line was employed to determine the extent to which recombinant human (rh) forms of the pro-inflammatory cytokines, tumor necrosis factor-α (rhTNF-α,), interleukin-6 (rhIL-6) and oncostatin M (rhOSM) induced apoptosis., Methods: The induction of apoptosis in the presence or absence of these cytokines was measured by the DAPI/TUNEL assay, by whether or not pro-caspase-3 was activated and by the extent to which poly-ADP-ribose polymerase (PARP) was degraded., Findings: Only rhTNF-α, and rhIL-6 significantly increased apoptosis in C-28/I2 chondrocytes, although rhOSM exhibited a strong trend (p=0.067) towards increasing the frequency of apoptotic chondrocytes. The number of apoptotic C28/I2 chondrocytes was significantly increased (p=1.3 × 10-5) by the combination of rhTNF-α and U0126 (10 μM) compared to rhTNF-α alone. However apoptosis was not further increased by combining rhIL-6 with U0126. The LI-COR ® western blot system showed that U0126 (10 μM) inhibited the phosphorylation of extracellular signal-regulated kinase-2 (p-ERK2) by phorbol myristate acetate-treated immortalized myometrial cells, U0126 (10 μM) did not alter total U-ERK2. Western blot analysis also revealed that the increased frequency of apoptotic C-28/I2 chondrocytes induced by rhTNF-α and rhOSM, but not rhIL-6, was associated with PARP degradation. However, none of the cytokines resulted in pro-caspase-3 activation., Conclusion: These results showed that rhTNF-α and rhIL-6 were strong inducers of apoptosis in the immortalized C-28/I2 human chondrocyte cell line. They also suggested that inhibiting ERK2 phosphorylation via U0126-mediated inhibition of MEK1/2 activity, increased rhTNF-α-induced C-28/I2 chondrocyte apoptosis.

The non-linear optical effect known as second harmonic generation (SHG) has been recognized since the earliest days of the laser. But it has only been in the last 20 years that it has begun to emerge as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. This is because only small modifications are required to equip a standard laser scanning 2-photon microscope for second harmonic imaging microscopy (SHIM). SHG signals from certain membrane-bound dyes are highly sensitive to membrane potential, indicating that SHIM may become a valuable probe of cell physiology. However, for the current generation of dyes and microscopes, the small signal size limits the number of photons that can be collected during the course of a fast action potential. Better dyes and optimized microscope optics could ultimately lead to the ability to image neuronal electrical activity with SHIM.

Introduction: Neuromyelitis optica (NMO) is characterized by inflammatory demyelinating lesions of the spinal cord and optic nerves from an autoimmune response against water channel aquaporin-4 (AQP4). We report 2 patients with transient hyperCKemia associated with NMO suggesting possible skeletal muscle damage., Methods: Patient 1 was a 72-year-old man who presented with muscle soreness and elevated serum creatine kinase (CK) preceding an initial attack of NMO. Patient 2 was a 25-year-old woman with an established diagnosis of NMO who presented with diffuse myalgias, proximal upper extremity weakness, and hyperCKemia. Muscle biopsies were obtained for histopathologic evaluation, protein gel electrophoresis, immunofluorescence, and complement staining., Results: In both patients the muscle showed only mild variation in fiber diameter. There were no inflammatory changes or muscle fiber necrosis, though there was reduced AQP4 expression and deposition of activated complement., Conclusions: Complement-mediated sarcolemmal injury may lead to hyperCKemia in NMO., (© 2014 Wiley Periodicals, Inc.)

Multiplex automated genome engineering (MAGE) is a powerful technology for in vivo genome editing that uses synthetic single-stranded DNA (ssDNA) to introduce targeted modifications directly into the Escherichia coli chromosome. MAGE is a cyclical process that involves transformation of ssDNA (by electroporation) followed by outgrowth, during which bacteriophage homologous recombination proteins mediate annealing of ssDNAs to their genomic targets. By iteratively introducing libraries of mutagenic ssDNAs targeting multiple sites, MAGE can generate combinatorial genetic diversity in a cell population. Alternatively, MAGE can introduce precise mutant alleles at many loci for genome-wide editing or for recoding projects that are not possible with other methods. In recent technological advances, MAGE has been improved by strain modifications and selection techniques that enhance allelic replacement. This protocol describes the manual execution of MAGE wherein each cycle takes ≈ 2.5 h, which, if carried out by two people, allows ≈ 10 continuous cycles of MAGE-based mutagenesis per day.

The management of acute pancreatitis has seen many advances over the past three decades. Attempts to improve care have led to new definitions, classification systems, and treatment strategies. Despite those efforts, considerable morbidity and mortality result from complications of severe acute pancreatitis. Much attention has been given to new ways to treat these complications, including inflammatory pancreatic fluid collections and associated infections. Endoscopy has become one of the established modalities for the treatment of these complications in many expert centers. This chapter will specifically address the role of endoscopy in the management of pancreatic necrosis.

This protocol describes the nonlinear optical phenomenon known as second-harmonic generation (SHG) and discusses its special attributes for imaging membrane-potential changes in single cells and multicellular preparations. Undifferentiated N1E-115 mouse neuroblastoma cells are used as a model cellular system for membrane electrophysiology. Styryl and naphthylstyryl dyes, also known as hemicyanines, are a class of electrochromic membrane-staining probes that have been used to monitor membrane potential by fluorescence; they also produce SHG images of cell membranes with SHG intensities that are sensitive to voltage. These experiments allow for the precise characterization of the voltage sensitivity of SHG and identification of the optimal wavelength for the incident laser fundamental light. This protocol presents the steps for the culture, staining, patching, and imaging of cells. The details of the imaging system and the measurements obtained are discussed, as are the prospects of this technology for imaging membrane potential changes in neuronal preparations., (© 2014 Cold Spring Harbor Laboratory Press.)

Posterior capsule opacification (PCO) is a debilitating and relatively common complication of cataract surgery despite modern medical and surgical advances. The pathophysiology of this condition has largely been attributed to peptide mediators, such as transforming growth factor-beta (TGFβ), epidermal growth factor (EGF) and matrix metalloproteinases (MMPs), with the inhibition of these and other related molecules showing promising results. Studies have also shown that the levels of interleukin-6 are elevated in the eyes following cataract surgery and in various ocular inflammatory disorders that predispose to PCO development. This review proposes, utilizing ocular and extra-ocular disease models, several potential pathways through which IL-6 may modulate the activity of TGFβ, EGF, MMP-2/-9 and immune cells to promote PCO. Among the IL-6-mediated pathways discussed are the transactivation of EGF receptor, the up-regulation of TGFβ and MMP-2/-9, and the loss of ocular immune privilege through trans-signaling, down-regulation of T-regulatory cells (Tregs) and up-regulation of Th17 cells. After establishing both the potential role of ocular IL-6 in the development of PCO and the apparent upstream activity of IL-6 in relation to the known mediators of PCO, the author hypothesizes that intraoperative anti-IL-6 therapy may be of great benefit in reducing all parameters of PCO pathogenesis. This review also provides a strong basis for carrying out multiple experimental studies with IL-6 inhibitors to further elucidate the specific pathways by which IL-6 may promote PCO as well as to better determine what role inflammation may play in this disease process., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Quality measures for prognostication of colon cancer include the removal of 12 or more lymph nodes during colon resection. The purpose of this study was to determine whether emergent surgery is associated with inadequate lymph node harvest. The National Cancer Database (NCDB) was queried for colon cancer patients operated on at Huntington Memorial Hospital, Pasadena, California, from 2005 to 2010. Demographic data, indication for surgery, surgeon, stage, lymph node harvest, tumor location, method of surgery, chemotherapy use, and survival were recorded. Univariate analyses were performed to compare lymph node harvest with the variables listed. Three hundred fifty-three patients underwent colon resection between 2005 and 2010. Two hundred ninety-six patients with Stage I to III disease underwent 253 elective (85%) and 43 emergent (15%) colectomies. There was no statistical difference between rates of adequate lymph node harvest in emergent and elective patient groups (86.0 vs 88.1%, P=0.7). Inferior long-term survival was associated with emergent indication and inferior lymph node harvest. Lymph node harvest adequacy showed a gradual increase over time from 79.5 per cent in 2005 to 95.5 per cent in 2010. Despite a perception that emergent surgery is associated with inadequate lymphadenectomy, 5-year data from Huntington Memorial Hospital participation in NCDB does not suggest inferior lymph node harvests in patients operated on for obstruction or perforation.

Objective: PET/CT with (18)F-FDG shows metabolically active disease and is widely used for the diagnosis and follow-up of patients with cancer. Nonmetabolically active renal pathology may be missed without close attention to the CT portion of the study, whereas metabolically active pathology may be missed on PET because of physiologic tracer excretion in the kidneys. This article illustrates primary lesions of the kidney on FDG PET/CT with emphasis on key anatomic features and the appearance of tracer uptake., Conclusion: Close attention to both the FDG PET and CT portions of the study is essential to interpret renal pathology correctly on FDG PET/CT examinations.

Objective: PET/CT with (18)F-FDG is a powerful tool to evaluate patients with hematologic malignancy or to assess the burden of metastatic disease from solid tumors. Metabolically active renal pathology associated with lymphoma, leukemia, or metastatic disease can be missed without close attention to both the PET and CT portions of the study because of physiologic FDG excretion in the kidneys. This article illustrates the appearance of tracer uptake and the key anatomic features of lymphoma, leukemia, and metastatic disease involving the kidney on FDG PET/CT., Conclusion: Close attention to both the FDG PET and CT portions of an FDG PET/CT study is essential to evaluate the kidneys in oncology patients.

This paper uses advances in the ultrafast manipulation of light to address a general need in medicine for a clinical approach that can provide a solution to a variety of disorders requiring subsurface tissue manipulation with ultralow collateral damage. Examples are age-related macular degeneration (AMD), fungal infections, tumors surrounded by overlying tissue, cataracts, etc. Although lasers have revolutionized the use of light in clinical settings, most lasers employed in medicine cannot address such problems of depth-selective tissue manipulation. This arises from the fact that they are mostly based on one photon based laser tissue interactions that provide a cone of excitation where the energy density is sufficiently high to excite heat or fluorescence in the entire cone. Thus, it is difficult to excite a specific depth of a tissue without affecting the overlying surface. However, the advent of femtosecond (fs) lasers has caused a revolution in multiphoton microscopy (Zipfel et al. Nat. Biotechnol. 2003, 21, 1369-1377; Denk et al. Science 1990, 248, 73-76) and fabrication (Kawata et al. Nature 2001, 412, 697-698). With such lasers, the photon energy density is only high enough for multiphoton processes in the focal volume, and this opens a new direction to address subsurface tissue manipulation. Here we show in an AMD animal model, Ccr2 KO knockout mutant mice, noninvasive, selective fs two-photon photobleaching of pigments associated with AMD that accumulate under and in ultraclose proximity to the overlying retina. Pathological evidence is presented that indicates the lack of collateral damage to the overlying retina or other surrounding tissue.

An object is characterized by its amplitude and phase. However, when acquiring optical data about such an object, using a recording medium such as a camera, phase information is lost. Crystallography experienced a breakthrough in phase retrieval for large molecular entities by Max Perutz's introduction of "heavy atoms" using the method of isomorphous replacement. The availability of scanning probe microscopy and its full integration with optical microscopy allows us to apply these X-ray concepts to implement "heavy atom" restoration of phase in optical phase retrieval. In analogy to the heavy atom method, we acquire Fourier intensities in place of an X-ray diffraction pattern, and in place of the heavy atom, we utilize a nanometrically translatable point source of light based on the propagating field of a cantilevered near-field scanning optical microscopic (NSOM) probe controlled by an atomic force microscope (AFM). This integration of NSOM/AFM technology with far-field imaging achieves robust phase retrieval independent of external parameters, leading to 3D optical imaging. The methodology has super-resolution potential, and thus, heavy atom restoration of phase with super-resolution (HARPS) shows the potential of transparently integrated scanning probe microscopy with optical and other imaging modalities such as electron or ion optical imaging., (© 2011 American Chemical Society)

Second-harmonic generation (SHG) by membrane-incorporated probes is a nonlinear optical signal that is voltage-sensitive and the basis of a sensitive method for imaging membrane potential. The voltage dependence of SHG by four different probes, three retinoids (all-trans retinal), and two new retinal analogs, 3-methyl-7-(4'-dimethylamino-phenyl)-2,4,6-heptatrienal (AR-3) and 3,7-dimethyl-9-(4'-dimethylamino-phenyl)-2,4,6,8-nonatetraenal (AR-4), and a styryl dye (FM4-64), were compared in HEK-293 cells. Results were analyzed by fitting data with an expression based on an electrooptic mechanism for SHG, which depends on the complex-valued first- and second-order nonlinear electric susceptibilities (χ² and χ³) of the probe. This gave values for the voltage sensitivity at the cell's resting potential, the voltage where the SHG is minimal, and the amplitude of the signal at that voltage for each of the four compounds. These measures show that χ² and χ³ are complex numbers for all compounds except all-trans retinal, consistent with the proximities of excitation and/or emission wavelengths to molecular resonances. Estimates of probe orientation and location in the membrane electric field show that, for the far-from-resonance case, the shot noise-limited signal/noise ratio depends on the location of the probe in the membrane, and on χ³ but not on χ²., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Onychomycosis is caused by dermatophyte infection of the nail. Though laser energy has been shown to eliminate dermatophytes in vitro, direct laser elimination of onychomycosis is not successful due to difficulties in selectively delivering laser energy to the deeper levels of the nail plate without collateral damage. Femtosecond (fsec) infrared titanium sapphire lasers circumvent this problem by the nonlinear interactions of these lasers with biological media. This quality, combined with the deeply penetrating nature of the near-infrared radiation, allows elimination of deeply seeded nail dermatopytes without associated collateral damage. Nail cuttings obtained from patients with onychomycosis caused by Trichophyton rubrum underwent fsec laser irradiation using increasing laser intensities with the focus scanned throughout the whole thickness of the nail specimen. The efficacy of the laser treatment was evaluated by subculture. Scanning electron microscopy was used to determine fsec laser-induced collateral damage. We found that a fsec laser fluence of 7 x 10(31) photons m(-2) s(-1) or above successfully inhibited the growth of the fungus in all samples examined, whereas laser intensities above 1.7 x 10(32) photons m(-2) s(-1) affected the structure of the nail plate. Our findings suggest that T. rubrum-mediated onychomycosis may be treated by fsec laser technology.

The general nanoprinting and nanoinjection of proteins on non-conducting or conducting substrates with a high degree of control both in terms of positional and timing accuracy is an important goal that could impact diverse fields from biotechnology (protein chips) to molecular electronics and from fundamental studies in cell biology to nanophotonics. In this paper, we combine capillary electrophoresis (CE), a separation method with considerable control of protein movement, with the unparalleled positional accuracy of an atomic force microscope (AFM). This combination provides the ability to electrophoretically or electroosmotically correlate the timing of protein migration with AFM control of the protein deposition at a high concentration in defined locations and highly confined volumes estimated to be 2 al. Electrical control of bovine serum albumin printing on standard protein-spotting glass substrates is demonstrated. For this advance, fountain pen nanolithography (FPN) that uses cantilevered glass-tapered capillaries is amended with the placement of electrodes on the nanopipette itself. This results in imposed voltages that are three orders of magnitude less than what is normally used in capillary electrophoresis. The development of atomic-force-controlled capillary electrophoretic printing (ACCEP) has the potential for electrophoretic separation, with high resolution, both in time and in space. The large voltage drop at the tip of the tapered nanopipettes allows for significant increases in concentration of protein in the small printed volumes. All of these attributes combine to suggest that this methodology should have a significant impact in science and technology.

The first experimental results of the nonresonant second harmonic generation (SHG) studies of human erythrocytes membrane exposed to various glucose concentrations in phosphate buffered saline (PBS solution) are presented in this article. It is shown that the SHG signal from the membrane can be altered as a function of glucose concentration. The link between the variation of the SHG intensity and the membrane dielectric permittivity with glucose is established both theoretically and experimentally by comparison with time domain dielectric spectroscopy (TDDS) measurement data.

Sulfotransferase 4A1 (SULT4A1) is a novel sulfotransferase expressed almost exclusively in the brain. The gene is located on chromosome 22q13.3, a region implicated in predisposition to schizophrenia. Recently, a variable microsatellite region located upstream of SULT4A1 was found to be associated with an increase in schizophrenia risk. We hypothesised that if functional dysregulation of SULT4A1 was involved in the aetiology of schizophrenia, then genetic variants in the coding sequence of SULT4A1 might be identified in cases compared with controls. To test this, we carried out a mutation analysis of the coding region (exons 2-7) in 71 Australian schizophrenia cases and 69 controls. We found no mutations, either synonymous or nonsynonymous, in either cohort. However, intronic variants (IVS5+12 C>T and IVS5+28 G>C) were identified, the frequency of which was not statistically different between cases and controls. The lack of polymorphisms in the coding region of the SULT4A1 gene is highly unusual and, along with its high conservation between species, suggests that SULT4A1 may have an important function in vivo. However, our findings do not support the hypothesis that germline mutations in the coding region of SULT4A1 contribute to susceptibility to schizophrenia.

In this review, we highlight the physical and enzymatic properties of the novel human sulfotransferase, SULT4A1. The gene is most highly expressed in selective regions of the brain, although work to date has failed to identify any specific endogenous substrate for the enzyme. SULT4A1 shares low homology with other human sulfotransferases. Nevertheless, it is highly conserved between species. Despite the low homology, it is structurally very similar to other cytosolic sulfotransferases with a conserved substrate binding domain, dimerization site and partial cofactor binding sites. However, the catalytic cavity is much smaller, and it has been suggested that the cofactor may not be accommodated within it. A recent link between variability in the 5'UTR of the SULT4A1 gene and schizophrenia has heightened interest in the endogenous function of the enzyme and its possible role in human disease.

Most of the known breast cancer susceptibility genes (BRCA1, BRCA2, CHEK2 and ATM) are involved in the damage response pathway. Other members of this pathway are therefore good candidates for additional breast cancer susceptibility genes. ATR, along with ATM, plays a central role in DNA damage recognition and Chk1 relays checkpoint signals from both ATR and ATM. PPP2R1B and PPP2R5B code for subunits of protein phosphatase 2A (PP2A), which regulates autophosphorylation of ATM. In addition, EIF2S6/Int-6, which was originally identified as a common integration site for the mouse mammary tumour virus in virally induced mouse mammary tumours, is a candidate breast cancer susceptibility gene because of its putative role in maintaining chromosome stability. To investigate the role of ATR, CHK1, PPP2R1B, PPP2R5B and EIF2S6/Int-6, we carried out mutation analysis of these genes in the index cases from non-BRCA1/BRCA2 breast cancer families. We also screened sporadic breast tumours for somatic mutations in PPP2R1B and PPP2R5B. Although we identified many novel variants, we found no evidence that highly penetrant germline mutations in these five genes contribute to familial breast cancer susceptibility.

A femtosecond laser beam gene transduction (SG-LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 microg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg-specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNgamma, and IL-4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG-LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV-infected replicating cells. Tumor growth retardation was induced in vaccinated mice challenged with an HBsAg-expressing syngeneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.

Extra ordinary transmission through arrays of subwavelength apertures has been investigated using near-field scanning optical microscopy. For such studies arrays were fabricated to give maximum resonance enhancement of light transmission at the wavelength of illumination that was used (532 nm). To define this enhancement a design was employed that allowed in one field of view of a near-field image the investigation of single apertures of dimension that was similar to what was incorporated into the sub-wavelength hole array. Significant asymmetry in the transmission and the propagation of the light along the aperture array was detected. This non-uniformity could be explained by polarization of the incident light, edge effects and the geometry of the array. The results support a hypothesis of both enhanced transmission due to surface plasmons and a non-diffracting beaming as a function of distance effect in the propagation of the light from the array.