Mechanism of an intramembrane chaperone for multipass membrane proteins.

Multipass membrane proteins play numerous roles in biology and include receptors, transporters, ion channels and enzymes1,2. How multipass proteins are co-translationally inserted and folded at the endoplasmic reticulum is not well understood2. 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 channel3-9. The PAT complex, an intramembrane chaperone comprising Asterix and CCDC47, engages early TMDs of multipass proteins to promote their biogenesis by an unknown mechanism10. 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 cavity11. 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.

Safe implementation of a minimally invasive hepatopancreatobiliary program, a narrative review and institutional experience.

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.

A unified evolutionary origin for the ubiquitous protein transporters SecY and YidC.

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.

Resection of the Primary Gastrointestinal Neuroendocrine Tumor Improves Survival With or Without Liver Treatment.

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.

Micrometer to 15 nm Printing of Metallic Inks with Fountain Pen Nanolithography.

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.

Wireless Communication with Nanoplasmonic Data Carriers: Macroscale Propagation of Nanophotonic Plasmon Polaritons Probed by Near-Field Nanoimaging.

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.

Live cell near-field optical imaging and voltage sensing with ultrasensitive force control.

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.

Pancreatic Neuroendocrine Tumors: State-of-the-Art Diagnosis and Management.

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.

Minimally invasive distal pancreatectomy: greatest benefit for the frail.

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.

Nanodrawing of Aligned Single Carbon Nanotubes with a Nanopen.

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.

Timing and severity of post-discharge morbidity after hepatectomy.

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.

Radioembolization following Liver Resection: Safety and Dosing Considerations.

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.

The New Face of Head and Neck Cancer: The HPV Epidemic.

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.

Second Harmonic Imaging of Membrane Potential.

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.

Transient hyperckemia in the setting of neuromyelitis optica (NMO).

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.

The role of endoscopy in the management of pancreatic necrosis.

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.

Structural analysis of the dynamic ribosome-translocon complex.

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.

Evaluating Cationic Nanoparticles as Carriers of Antiviral Nucleic Acids.

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.

Host Associations of Culicoides Biting Midges in Northeastern Kansas, USA.

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.