Electroencephalogram (EEG) power reductions in the aging brain have been described by numerous previous studies. However, the underlying mechanism for the observed brain signal power reduction remains unclear. One possible cause for reduced EEG signals in elderly subjects might be the increased distance from the primary neural electrical currents on the cortex to the scalp electrodes as the result of cortical atrophies. While brain shrinkage itself reflects age-related neurological changes, the effects of changes in the distribution of electrical conductivity are often not distinguished from altered neural activity when interpreting EEG power reductions. To address this ambiguity, we employed EEG forward models to investigate whether brain shrinkage is a major factor for the signal attenuation in the aging brain. We simulated brain shrinkage in spherical and realistic brain models and found that changes in the conductor geometry cannot fully account for the EEG power reductions even when the brain was shrunk to unrealistic sizes. Our results quantify the extent of power reductions from brain shrinkage and pave the way for more accurate inferences about deficient neural activity and circuit integrity based on EEG power reductions in the aging population. Decreases in scalp EEG amplitude have been described previously in normal aging Polich,  and in pThe striking reductions in EEG power during aging and AD could be explained by a number of factors. One interpretation would be that decreases in EEG power reflect underlying reductions in cortical current amplitudes due to reduced synaptic density, activity, synchronization, or some combination therein  and a fast low-angle shot gradient echo sequence  using a 12-channel RF receive coil array on a 3T Siemens TrioTM MR scanner. An equidistant 128-channel montage  was used for EEG electrode placement, and electrode positions were digitized using the Polhemus FastSCAN II laser scanner, aligned to the subject's structural MRI based on fiducial points (nasion and preauricular points) using the iterative closest point algorithm, and projected onto the scalp surface.Structural MRI scans were collected from one healthy young subject at Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital. The subject gave written informed consent, and the study protocol was approved by the Massachusetts General Hospital Human Research Committee. Brain structural anatomical details were obtained from a T1-weighted multi-echo sequence , and “mri2mesh” was repeated with this corrected pial surface to provide an improved, updated tissue segmentation.In the four-layer MRI-based realistic volume conductor models, we computed the electromagnetic field using the BEM solutions to approximate boundary surface potentials. Tissue segmentation and mesh construction of boundary surfaces were initialized by running two SimNIBS processing pipelines using default settings: (1) “mri2mesh” based on a FreeSurfer 6.0 reconstruction  surfaces were obtained from “mri2mesh.” For the outer skull surface, CAT12 within the “headreco” pipeline provided more accurate segmentation of bone structures compared to “mri2mesh.” However, “headreco” outlined facial bone structures that were too detailed than appropriate for BEM solutions. Therefore, we constructed a hybrid outer skull surface combining the superior and posterior quadrants obtained with “headreco” and the anterior-inferior  quadrant obtained using “mri2mesh”  acceleration have been developed  closest to the scalp EEG electrode was activated, while keeping the other sources silent. Each dipole in the active patch was assumed to have the same amplitude. The power reduction for each shrinkage value was calculated directly from the forward model gain matrices (see We simulated the overall structural effect of brain atrophy by shrinking the radius of the brain while maintaining the dimensions of the other tissue layers, i.e., skull and scalp. This “brain shrinkage” moves cortical dipoles away from the scalp and expands the CSF layer, in a manner analogous to age-related brain atrophy. This whole-brain shrinkage was implemented by reducing the distance from each source to the median centroid of all source dipoles by varying percentages of 0-30% in increments of 5% in both the spherical and realistic MRI-based models. We maintained the same number of dipoles across all levels of shrinking. For the spherical models, due to symmetry, only a single electrode was needed to characterize power reductions measured on the scalp . To simuIn the realistic models, simulated shrinkage affected different cortical regions slightly differently due to anatomical variations and non-spherical geometry. The pial surface and the dipole locations were shrunk over the same range of percentages, 0-30% in 5% increments, to model the expansion of the CSF layer under cortical atrophy . We estiAs described earlier, we set the tissue conductivities in our models to values at the midpoints of the published ranges. However, multiple studies have highlighted how uncertainties in skull and CSF conductivities can impact on the accuracy of forward models (Gençer and Acar, In this study we analyzed the extent to which age-dependent EEG signal power reductions could be attributed to structural changes in the brain. Using both spherical and realistic MRI-based forward models, we found that physiologically plausible levels of cortical atrophy produced only modest decreases in EEG power, which are small compared to those seen in aging and age-related dementia (Babiloni et al., A critical component of our approach was to use four-layer BEM forward models to provide anatomically accurate simulations of shrinking cerebral volume that could incorporate the corresponding increase in CSF volume. This approach requires accurate extraction of tissue boundary meshes from MRI scans. We devised a novel workflow combining three different MRI mesh-extraction pipelines in FreeSurfer, SimNIBS, and MNE-Python to take advantage of the best properties of these respective methods. We also analyzed four-layer spherical models and observed EEG power reductions that were highly comparable to those observed using the realistic models. Corroborating results from the spherical and realistic models together provided consistent estimates of EEG power reductions from structural brain atrophy in aging. Potential age-related changes in the skull and skin thickness were not evaluated in this study after careful consideration. The skull thickness does not change appreciably with advancing age beyond early adulthood except in cases of specific pathology (Albert et al., Our simulations modeled cerebral atrophy as a “shrinkage” in the brain parenchymal volume while holding other tissue boundaries constant. This simulation procedure captures three different effects of real brain atrophy. First, a primary effect of the shrinkage was to increase the thickness of the CSF layer. As shown in In our simulations, the CSF layer expanded by more than three-fold from a starting point of 2.9 mm average thickness at 0% shrinkage, to 5.8 and 8.9 mm at 5 and 10% shrinkage, respectively. The latter value is comparable to the CSF thicknesses observed in severe AD (Ancora et al., An important caveat of the simulation analyses is that the inter-subject variability of anatomical details including the extent of cortical atrophy is not modeled. Our approach combines simulated shrinkage using realistic models based on a single MRI and generalizable spherical models with results that are highly consistent. Power reductions are quantified in this study similar to a longitudinal study design, while empirically observed age-related EEG power changes are cross-sectional comparisons with individual differences contributing to the level of power reductions observed. Additional factors such as cortical folding and scalp location when measuring EEG signals can also influence the absolute scales of power reductions . Our simAside from the modest influence on volume conduction investigated here, age-related decreases in EEG power can also reflect an overall reduction in the size of cerebral currents. This could occur through a variety of mechanisms, including reductions in post-synaptic current amplitude, neuronal density, and/or neuronal synchrony, all of which have significant functional and pathophysiologic implications. Overall, head anatomy varies substantially between individuals and across age ranges. Realistic MRI-based forward models could be employed to gauge the individualized impact of structural changes on scalp EEG signals, to facilitate accurate interpretations of experimental findings of EEG power reductions, and to maximize the diagnostic precision of EEG-based biomarkers for age-related neurological disorders. Our results suggest that while structural changes due to brain atrophy play a role, age-dependent attenuation of the EEG signal more likely reflects underlying functional and neurophysiological changes that are fundamental characteristics of aging and age-related disease.The raw data supporting the conclusions of this article will be made available by the authors upon request, without undue reservation.The studies involving human participants were reviewed and approved by Massachusetts General Hospital/Brigham and Women's Hospital Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.MH: formal analysis, software, data curation, and first draft writing. FL: data curation, formal analysis, software, and review-editing. AN: software, supervision, and review-editing. MH: review-editing and supervision. BD: review-editing. PP: grant acquisition, supervision, conception and review-editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Helium ion beam induced deposition using the gaseous precursor pentamethylcyclopentasiloxane is employed to fabricate high aspect ratio insulator nanostructures (nanopillars and nanocylinders) that exhibit charge induced branching. The branched nanostructures are analyzed by transmission electron microscopy. It is found that the side branches form above a certain threshold height and that by increasing the flow rate of the precursor, the vertical growth rate and branching phenomenon can be significantly enhanced, with fractalesque branching patterns observed. The direct-write ion beam nanofabrication technique described herein offers a fast single-step method for the growth of high aspect ratio branched nanostructures with site-selective placement on the nanometer scale. Focused ion beams (FIB) are widely used for additive and subtractive nanofabrication, either via direct-write exposure, or using resist-based methods. In the direct-write approaches, the ion beam locally removes material by sputtering, or adds material by FIB-induced deposition (FIBID) . In the For the deposition of electrically insulating structures, it is advantageous to use a non-metallic ion species, since ions are co-implanted with the deposited material and can thus affect the resistivity. Furthermore, if deposits with the smallest critical dimensions are required, then the GFIS-based instrument is an ideal choice, since it delivers a probe size down to 0.5 nm and produces minimized feature broadening due to the small interaction volume of the light ions near the sample surface . To dateIn the early helium FIBID nanopillar studies mentioned above , it was In this study, helium FIBID of high aspect ratio nanostructures (nanopillars and nanocylinders) using the insulator precursor pentamethylcyclopentasiloxane (PMCPS) is investigated, with particular focus on characterizing the resulting branched structures by TEM and investigating growth rate effects. The nanostructures are grown directly onto TEM-compatible substrates to allow direct imaging and elemental analysis at high spatial resolution without the need for further sample preparation steps. It is found that, by increasing the flow rate of the precursor, the vertical growth rate can be significantly enhanced and more complex branching morphologies are obtained.The deposition experiments were performed using a Zeiss ORION NanoFab helium/neon/gallium FIB microscope , which incorporates a GFIS column for helium and neon FIB, and a conventional gallium FIB column. The GFIS was operated with a helium gas pressure of 1–5 × 10NanoPatterning and Visualization Engine (NPVE) software from Fibics, Inc.  was used to control the beam during the FIBID process, selecting ‘spot mode’ exposures to grow the nanopillars  and 3 mm silicon TEM half-grids (Dune Sciences). The copper grids were cut in two along the length of the slot, using a sharp razor blade to produce half-grids similar in shape to the silicon half-grids. The half-grids were mounted onto aluminum low-profile 36After the depositions, low-magnification images of the deposits were first acquired by helium ion microscopy TEM results for PMCPS nanopillars and nanocylinders grown using a higher precursor flow rate .The results for a series of PMCPS nanocylinders also grown using the higher precursor flow rate are presented in The PMCPS nanocylinder that is pictured in Notably, in related FEBID experiments concentrating on PMCPS nanopillars fabricated using the electron beam of the SEM, de Boer et al. observed a large enhancement in branching when changing from a bulk silicon substrate (semiconducting) to a silicon nitride membrane (insulating) . As mentIn order to investigate the growth rates in more detail, plots of nanostructure height versus deposition time are shown in For the PMCPS nanopillars grown using the ‘standard’ precursor flow rate lsewhere ,19,21, bA representative TEM image of a tungsten-based nanopillar deposited using the highest dose (deposition time 16 s) is shown in The widths of the tungsten-based nanopillars were measured at ∼30–40 nm, irrespective of the substrate and in agreement with the widths of metallic (platinum and tungsten) helium FIBID nanopillars reported in the literature ,21,23,24This study has shown that insulating high aspect ratio nanostructures grown by helium FIBID exhibit a branching phenomenon attributed to charging that is not observed for their metallic counterparts. Branching occurs above a certain threshold height and branching behavior can be significantly enhanced by increasing the flow rate of the gaseous precursor, leading to faster growth and the formation of complex fractal-like branching patterns. Diffraction analysis of the branch structures reveals a nanocrystalline component in the otherwise amorphous material. For applications requiring high aspect ratio nanopillars with smooth sidewalls, charge-neutralization strategies may be applied. However, other applications may directly benefit from branched nanopillars such as those demonstrated here, which can be deposited efficiently at precisely defined locations in a single-step process using the helium FIBID method.


Goal-directed fluid therapy optimizes cardiac performance and thus tissue perfusion and oxygen delivery. We therefore tested the hypothesis that intra- and postoperative PsqO2 is significantly reduced in obese patients undergoing standard fluid management compared to goal-directed fluid administration.Perioperative subcutaneous tissue oxygen tension (PsqO2) undergoing laparoscopic bariatric surgery to receive either esophageal Doppler-guided goal-directed fluid management or conventional fluid treatment. Our primary outcome parameter was intra- and postoperative PsqO2 measured with a polarographic electrode in the subcutaneous tissue of the upper arm. A random effects linear regression model was used to analyze the effect of intervention.We randomly assigned 60 obese patients . No effect was seen intraoperatively ; however, goal-directed fluid management improved PsqO2 in the early postoperative phase . Intraoperative fluid requirements did not differ between the two groups.Overall, mean (± SD) PsqOGoal-directed fluid therapy improved subcutaneous tissue oxygenation in obese patients. This effect was more pronounced in the early postoperative period.ClinicalTrials.gov (NCT 01052519).The study was registered at  None of2, is a major determinant of perioperative tissue oxygenation. Subcutaneous tissue and wound hypoxia are common in obese patients during the perioperative period [Obesity, defined as a BMI ≥ 30 kg/me period . Even wie period .2, which is far below the BMI range of the typical patient who is referred to weight loss surgery. The study presented here adds further weight to the evidence, as morbidly obese patients undergoing bariatric surgery with an average BMI of approximately 45 kg/m2 were included and randomly assigned to a treatment group and a conventional group.In this study, overall subcutaneous tissue oxygen tension increased significantly in obese patients undergoing laparoscopic surgery receiving Doppler-guided fluid administration. While no difference was observed intraoperatively, the effect of intervention resulted in raised tissue oxygenation in the early postoperative period. Our findings are in accordance with a recently published paper showing that subcutaneous tissue oxygen tension was high and well maintained in lean, overweight, and obese patients undergoing Doppler-guided fluid management during open gynecological surgery . HoweverWe have previously shown that subcutaneous tissue oxygenation in obese patients undergoing laparoscopic surgery was reduced by roughly 15 mmHg compared to non-obese patients . There iLaparoscopic case volumes have increased over time; especially in obese patients with an increased risk of wound healing complications, laparoscopic interventions represent state-of-the-art techniques. However, tissue oxygen tension is decreased during laparoscopic colonic surgery compared to open surgery . DuratioInterestingly, our observed improvement of tissue oxygen tension in obese patients assigned to the goal-directed group was not reflected by systemic hemodynamics, which remained unchanged between both groups. One may argue that goal-directed volume optimization has a more pronounced effect during open abdominal procedures. So far, only a few studies have focused on the coherence between optimized global hemodynamics and microcirculation during perioperative GDFT , 29. In Given the cardiovascular peculiarities of obese patients, the assumption of hemodynamic coherence, in the specific setting of pneumoperitoneum, remains questionable. Alongside macro- and microcirculation, arterial oxygenation constitutes a major determinate of tissue oxygen availability . One strThe use of vasopressors and subsequent vasoconstriction is an important confounding factor for tissue oxygen tension. However, phenylephrine use in our study was comparable in both arms and had no effect on tissue oxygen tension.2 from a needle-induced surrogate wound in the upper arm. This represents the classical method of measuring tissue oxygenation and has been validated and used in numerous previous studies [A limitation is that we did not measure gut or wound tissue oxygen tension but instead evaluated PsqO studies . The ben studies .All our patients were relatively healthy; obese patients were predominately female and procedures were short. The beneficial effect of guided stroke volume optimization might be more obvious in patients with cardiovascular compromise, in male patients with a higher degree of central obesity and during longer procedures with more extended fluid shifts. We did not expect any long-lasting effects of our fluid regimen, which was restricted to the intraoperative period. Thus, we only measured tissue oxygen tension during a short period of time in the immediate postoperative period. The first hours after surgical trauma are considered to represent a decisive period for the establishment of surgical site infection and are thus of major interest .Regardless of similar systemic hemodynamics and comparable fluid requirements, goal-directed fluid therapy improved subcutaneous tissue oxygen partial pressure in the early postoperative period in obese patients undergoing laparoscopic bariatric surgery. To enhance peripheral perfusion and thus tissue oxygen availability, the timely administration of fluid boluses according to patients’ needs might be more important than the total amount of fluid. Given the laparoscopic approach and the short duration of surgery, our findings might have more impact in patients undergoing open abdominal surgery associated with significant fluid shifts.


Salmonella enterica serovar Typhi strain in clinical trials in 1984, with excellent humoral immune responses against the carrier strain but only modest responses elicited against the foreign antigen. Since then, clinical trials with additional Salmonella-based carrier vaccines have been conducted. As with the original trial, only modest foreign antigen-specific immunity was achieved in most cases, despite the incorporation of incremental improvements in antigen expression technologies and carrier design over the years. In this review, we will attempt to deconstruct carrier vaccine immunogenicity in humans by examining the basis of bacterial immunity in the human gastrointestinal tract and how the gut detects and responds to pathogens versus benign commensal organisms. Carrier vaccine design will then be explored to determine the feasibility of retaining as many characteristics of a pathogen as possible to elicit robust carrier and foreign antigen-specific immunity, while avoiding over-stimulation of unacceptably reactogenic inflammatory responses.The use of live-attenuated bacterial vaccines as carriers for the mucosal delivery of foreign antigens to stimulate the mucosal immune system was first proposed over three decades ago. This novel strategy aimed to induce immunity against at least two distinct pathogens using a single bivalent carrier vaccine. It was first tested using a live-attenuated  Salmonella enterica-based carrier vaccines with an eye towards optimizing protective efficacy while maintaining acceptable reactogenicity. We will first summarize the state-of-the-art for clinical trials conducted using carrier vaccines derived from S. Typhi. We will then attempt to deconstruct the lack of carrier vaccine immunogenicity in humans by examining the basis of bacterial immunity in the human gastrointestinal tract and how the gut detects and responds to pathogens. Carrier vaccine design will then be explored to determine the feasibility of retaining as many characteristics of a pathogen as possible to elicit robust carrier and foreign-antigen-specific immunity, while avoiding overstimulation of unacceptably reactogenic inflammatory responses. The insights gleaned from this discussion will provide a basis for possible solutions that may significantly improve the overall immunogenicity of S. Typhi-based carrier vaccines in humans.The concept of bacterial-based carrier vaccines rests on the notion that an attenuated strain of bacteria, genetically engineered to be safe yet still immunogenic, can be further engineered by the introduction of additional genes encoding protective antigens from unrelated pathogens; the resulting multivalent candidate vaccine should then be capable of eliciting biologically relevant protective immune responses against both the carrier vaccine itself as well as the additional target pathogens. Although simple in principle and elegant in terms vaccine development, this novel approach has required refinement through various iterations over the last three decades, with testing in clinical trials yielding only limited success. In this review, we will re-examine the engineering of S. Typhi-based carrier vaccines have been tested in clinical trials, as summarized in Since 1984, at least a dozen S. Typhi-based carrier vaccines have focused on foreign-antigen-specific humoral immunity rather than antigen-specific, cell-mediated immunity. However, few of these trials demonstrated foreign-antigen-specific seroconversions in the majority of volunteers, with many studies reporting percentages well below 25%. A successful clinical trial described by Bumann et al. in 2010 [Pseudomonas aeruginosa; all volunteers were then boosted intramuscularly (i.m.) 4 weeks later with a single dose of alum-adjuvanted purified fusion protein. These vaccinees mounted P. aeruginosa-specific serum IgG responses comparable to subjects immunized with 3 i.m. doses of adjuvanted subunit vaccine alone; however, orally primed volunteers also mounted P. aeruginosa-specific mucosal pulmonary IgA responses that were not observed in subjects receiving only i.m. immunizations. Interestingly, in an additional cohort of volunteers vaccinated with live vector vaccines derived from the more attenuated licensed vaccine Ty21a, three oral priming doses in addition to the i.m. booster dose were required to elicit immune responses comparable to those of volunteers receiving only a single oral priming dose of CVD 908 plus i.m. boost [Salmonella-based carrier vaccines and the antigens that they co-express, we will briefly step back to examine a broader question of how the human intestine regulates immunity against pathogens versus non-pathogenic bacteria in the gut.Most reported clinical trials studying  in 2010  illustram. boost . AlthougThe gastrointestinal tract supports an extensive diversity of commensal bacteria, with the highest colonization densities residing in the colon ,15. The reg cells, thereby maintaining an important balance between immunosuppressive Tregs and effectors such as IL-17-secreting Th17 and Th1 effector cells to regulate inflammation [The metabolism of resident human microflora also provides metabolites to the host that are not normally available from human metabolic activity, including the short-chain fatty acids (SCFAs) butyrate, propionate, and acetate . SCFAs cammation ,21.The detailed mechanisms by which humans regulate inflammation and control mucosal immune responses against commensal bacteria to preserve homeostasis are not fully understood. The process of deciphering these pathways has been likened to the old parable of blind men in a room describing an elephant; descriptions will depend on where and what one is attempting to examine . HoweverPhysical containment of commensal bacteria to prevent unrestricted access to deeper tissue immune inductive sites is accomplished by mucus layers which overlay a single cell layer of IECs covering the lamina propria, where other immune inductive cells reside . A thin + Foxp3+ regulatory T cells (Tregs) to stimulatory CD4+ RORγt+ helper T cells (Th17 cells) [regs [regs, thereby maintaining antigen-specific, commensal-targeted homeostasis [regs expressing gut-homing α4β7 and CCR9 surface markers that facilitate maintenance of gut homeostasis [regs secrete TGF-β and IL-10 to suppress Th17 cells and inflammation [The balance between commensal homeostasis and the induction of inflammatory reactions, accompanied by subsequent adaptive responses against pathogenic organisms, depends on the ratio of CD47 cells) ,26,27,287 cells) ,29,30,31s) [regs . Dendrits) [regs  to bind s) [regs ,34. In meostasis ,36. Non-eostasis ,38. Thesammation .regs [regs [Despite the central importance of APCs in maintaining homeostasis versus adaptive immunity, there are several other subsets of intestinal cells that are important in distinguishing between non-pathogenic and pathogenic organisms. Perhaps not surprisingly, IECs play an important role in sensing the invasion of microbes. Flagellin appears to be one important signaling molecule which binds to TLR5. Importantly, TLR5 is expressed in the basolateral membrane of non-activated IECs rather than in the apical membrane, where the concentration of flagellin from commensal organisms would be highest ,40,41. Tregs , again ags [regs ,43.Another population of immune cells that play an important role in the maintenance of homeostasis are innate lymphoid cells (ILCs), which do not express high-affinity antigen receptors . ILC3s, It is clear then that the maintenance of a symbiotic relationship with non-pathogenic bacteria in the gastrointestinal tract is preserved by intricate pathways of cytokine communication between IECs, ILCs, macrophages, and dendritic cells, with redundancy built in to ensure proper segregation of normal flora from immune inductive sites. Breach of these barriers by pathogenic organisms, equipped with an array of virulence factors capable of defeating primary defenses against infection, triggers innate inflammatory responses and full activation of dendritic cells, which migrate to regional lymph nodes to elicit robust induction of adaptive immunity.In view of the preceding discussion, it becomes clear that one reason that pathogenic organisms trigger pathogen-specific adaptive immune responses involves the activation of inflammatory responses, which recruit innate immune cells such as systemic macrophages, neutrophils, and cytotoxic natural killer cells to the area of infection to rapidly begin phagocytosing and killing invading organisms while activating other antigen-presenting cells such as dendritic cells. The mechanisms controlling the level of immune response against a given bacterial organism depend on overlapping and complementary detection strategies capable of discriminating between non-pathogenic and pathogenic organisms to mount either a tolerogenic non-inflammatory response or to trigger more robust adaptive immunity, respectively. Indeed, a theory describing a series of immune checkpoints for scaling the threat of a microbial infringement has recently been advanced ,48, for Salmonella enterica serovar Typhimurium presented to the immune system either as particulate purified outer membrane vesicles (OMVs) or as soluble conjugate vaccines in which the O-antigen was chemically conjugated to the hapten carrier protein CRM197. Particulate OMVs were able to induce higher O-antigen-specific IgG responses with broader IgG subclass and Ig isotype profiles than soluble glycoconjugates, accompanied by strong bactericidal activity and reduced bacterial colonization, in mice challenged with virulent S. Typhimurium strains. Importantly, OMVs did not require the use of adjuvants while non-adjuvanted glycoconjugates were not immunogenic [These immune checkpoints can respond to five key properties of invading pathogens that have been postulated to influence the strength of an adaptive immune response through interaction with these checkpoints. The first property concerns the particulate nature of ligands associated with invading pathogens, which is proposed to signal a higher microbial threat to the host immune system than soluble ligands. This would imply that intact organisms possessing multiple cell-associated ligands to activate different pattern-recognition receptors (such as lipopolysaccharide activation of TLR4 or flagellin activation of TLR5) could result in robust activation of innate immune cells and subsequent pathogen-specific adaptive immunity by multiple signaling pathways . Intact unogenic .The second property of invading pathogens proposed to affect adaptive immunity is viability. Live organisms elicit more robust immune responses than dead organisms. In this respect, mRNA may be a key signaling molecule for the presence of live microbial threats because it is normally degraded very quickly and is not present in high levels in non-living organisms. In support of this possibility, over a decade ago, it was first observed that the immunogenicity of killed bacteria could be raised to the level elicited by live-attenuated bacteria, both administered parenterally in equal dosing schedules, by supplementation of killed vaccine with purified mRNA . Accumul, have been previously reported and will not be recapitulated here [Salmonella (including S. Typhi), expression of ancillary effector proteins such as SopB is capable of delaying inflammatory responses by interrupting the proper function of TLRs through interference with adapter proteins such as TIRAP, which is required to facilitate downstream signaling of activation pathways [Salmonella of avoiding early inflammatory responses that could delay or diminish infection and promote clearance by adaptive responses.The third property of invading pathogens affecting adaptive immunity concerns live organisms with pathogenic factors facilitating colonization and/or secretion of toxins, which elicit more robust inflammatory and adaptive immune responses than non-pathogenic organisms without these additional factors. Excellent summaries of pathogenic secretion systems and other virulence factors, specifically relating to enteric bacteriated here ,56,57,58pathways ,60. ThisBacteroides fragilis, an organism that displays both commensal and pathogenic behavior in the same human host , carry a unique pathogenicity island (BfPAI) encoding a potent metalloprotease enterotoxin called Bacteroides fragilis toxin, or BFT [The last two properties of invading pathogens affecting immune responses involve invasion of host barriers, frequently initiated when endothelial tissue is breached, which triggers inflammatory reactions. This activity appears to influence the maintenance of non-invasive commensals in the gastrointestinal tract through immune homeostasis, as discussed above. However, deep tissue penetration into normally sterile sites, in which homeostasis does not occur, provides robust signaling for inflammatory and adaptive responses. The impact of these two properties on inflammatory responses is clearly illustrated in the case of gastrointestinal colonization with man host A,B. Cert, or BFT . Despite, or BFT ,62. It h, or BFT . HoweverSalmonella vaccines in general and Salmonella-based carrier vaccines in particular.These five properties of invasive pathogens, proposed to stimulate immune checkpoints controlling innate and adaptive immunity, seem to explain why reactogenic live oral vaccines have historically generated the most highly immunogenic pathogen-specific immune responses, while over-attenuated candidate vaccine strains have not provided biologically relevant immunity. Vaccines able to induce sufficient inflammatory responses via multiple signaling pathways can elicit a robust adaptive immune response capable of conferring protection against disease, while over-attenuated strains may be quickly cleared by innate cells (such as non-inflammatory macrophages) without eliciting inflammation. However, achieving the proper balance between acceptable reactogenicity and protective immunogenicity has proven to be a very elusive goal for engineering appropriately attenuated In addition to the ability to stimulate innate immunity, other critically important factors influence the quality of an adaptive immune response against carrier vaccines and the foreign antigen(s) that they present to the immune system. Based on data from both murine intranasal animal models ,65,66 anS. Typhi-derived carrier vaccines, we reason that limited invasion of gastrointestinal tissue, facilitated by retention of multiple virulence factors of S. Typhi, will result in the vaccine reaching immune inductive sites in numbers sufficient to trigger an innate immune response that triggers adaptive immune responses, without high levels of replicating organisms precipitating more serious inflammatory reactions. This delicate balance between minimal reactogenicity through attenuation and robust immunogenicity can be achieved through chromosomal deletion of two types of genetic loci: 1) deletion of critical genes with pleiotropic effects on the expression of multiple factors essential to survival within the host, or 2) deletions which interrupt metabolic pathways to restrict the replication of vaccines within the host. Examples of carrier vaccines in which pleiotropic deletion mutations resulted in clinically acceptable attenuated vaccine candidates include deletions of phoP/phoQ  of volunteers receiving a single oral dose containing 107 viable organisms [htrA, a stress protein, prevented vaccinemia while maintaining robust humoral and cellular immune responses against the vaccine strain [We hypothesize that retention of as many native characteristics of a given pathogen as possible, to elicit robust carrier and foreign-antigen-specific immunity while avoiding over-stimulation of unacceptably reactogenic inflammatory responses, is essential to the development of an immunogenic carrier vaccine. In the case of , ssaV , and ht, , and h08-htrA; ,68); allCVD 908, ,69) and trains”, ,70). In rganisms ; introdue strain ,68.htrA and Ty21a. Ty21a is a licensed typhoid live vaccine derived from the wild-type clinical isolate Ty2 by treatment with a chemical mutagen, resulting in multiple attenuating chromosomal lesions [htrA is a genetically engineered vaccine candidate in which only three precise deletion mutations have been introduced into Ty2 [htrA and Ty21a carrier vaccines expressing the urease foreign antigen from Helicobacter pylori, three doses of the Ty21a carrier containing 1010 viable organisms were required to elicit urease-specific humoral immunity comparable to levels elicited by CVD 908-htrA administered in a single dose of 108 viable organisms [In contrast to under-attenuated vaccine strains, over-attenuation of vaccine strains can result in candidates that prove completely safe and non-reactogenic in subjects but also fail to elicit immunogenic responses to either the carrier itself or the foreign antigen . However lesions ,73; in cinto Ty2 . In clinrganisms . These drganisms . S. Typhi are fully capable of eliciting mucosal, humoral, and cellular responses after oral immunization in humans [Since the first clinical trials conducted with carrier vaccines in the 1980s ,3,4, sign humans ,79,80,81n humans ,83, inapn humans ,85,86,87n humans ,74,75. HthyA [asd [, and purB [Over-attenuation of carrier vaccines due to the metabolic burden associated with inappropriate expression of foreign antigens often occurs when these antigens are expressed from plasmids of high copy number . This pothyA ,9,10,12,hyA [asd , and purand purB  have beeand purB ,11; the Salmonella-based carrier vaccine into the surrounding milieu, is significantly more immunogenic than cytoplasmically expressed antigen that may aggregate into insoluble inclusion bodies or be degraded by proteases [Streptococcus pneumonia surface protein A (PspA). The authors of this study speculated that poor growth of the vaccine in vivo, perhaps due to hyperattenuation, may not have permitted sufficient synthesis of PspA to stimulate immunity, again emphasizing the strict requirement for the carrier vaccine to reach immune inductive sites in sufficient numbers to elicit biologically relevant immune responses [Yet another issue that must be grappled with is the stability of the foreign antigen once synthesized by the carrier vaccine. Data from murine models of immunogenicity strongly suggest that surface expressed antigen, or antigen secreted from a roteases . A numberoteases ,99,100, roteases . Althougesponses .Helicobacter pylori [Pseudomonas aeruginosa [thyA-stabilized expression plasmids with medium copy numbers as well, and subjects received three oral doses of ~1010 viable organisms per dose in both studies. However, for subjects immunized with Ty21a expressing UreAB, 0/9 volunteers developed antigen-specific IgG responses, although rises in cell-mediated immunity were noted. In contrast, 15/16 volunteers receiving Ty21a expressing OprF-OprI seroconverted. To further emphasize this point, 13/16 volunteers seroconverted after receiving three doses of 100-fold less viable CVD 908-htrA organisms  and developed antibodies against OprF-OprI expressed from the identical thyA-stabilized expression plasmid in the same study [Supposing that one is able to adjust stable but sufficient expression levels of a foreign antigen while avoiding over-attenuation, an additional property impacting on antigen-specific immunity is the inherent immunogenicity of the antigen itself. This parameter is clearly observed in clinical trials of carrier vaccines expressing either the urease virulence factor from r pylori ,10 or a ruginosa . Recognime study . These oSalmonella-based carrier vaccines [S. Typhi-based carrier strain itself, which could theoretically reduce the numbers of organisms reaching immune inductive sites. Indeed, in studies conducted by T.F. Meyer and colleagues [S. Typhi-specific immunity elicited by repeated dosing of volunteers with a carrier vaccine has minimal effects on foreign-antigen-specific immunity, even in cases where doses as high as 1010 viable counts are given. This observation also appears to be independent of the spacing between doses, wherein neither administration of three doses within an 8-day period [Salmonella-specific immunity; therefore, the strategy of multiple dosing to elicit optimum humoral or cellular immunity against foreign antigens appears to constitute a sound immunization strategy. This strategy becomes especially powerful in the context of first immunizing orally with one or more doses of S. Typhi carrier vaccine and then boosting parenterally with purified adjuvanted foreign antigen, an immunization regime referred to as heterologous prime-boosting and first explored for the improvement of malaria and HIV subunit vaccines [S. Typhi carrier vaccines has been shown in murine intranasal models of immunogenicity to elicit excellent antigen-specific immunity, often superior to immunity elicited from multiple doses of carrier vaccine alone [Clinical trials have proven essential for working out an effective immunization strategy, capable of eliciting robust and biologically relevant immunity against foreign antigens delivered to the immune system after oral inoculation. Of the trials conducted to date, both single and multiple dosing schedules have been tested , with sevaccines ,103,104,lleagues ,10, it wy period ,9,10,12 y period  exerted vaccines ,106. Hetne alone ,109,110.In addition to factors influencing the antigen-specific immunity of carrier vaccines, one must also consider factors which can significantly affect the immunocompetence of the population intended to receive a given carrier vaccine. Given the complexity of mechanisms influencing immune function, a number of host-specific determinants should also be expected to affect the quality and specificity of an immune response to a carrier vaccine. Although the age of the individual and comorbidity status are two important determinants currently receiving significant attention ;(1) the choice of an attenuated (2) expression from genetically stabilized multicopy expression plasmids of sufficient foreign antigen to elicit biologically relevant immunity (antigen expression factors);(3) use of a heterologous prime–boost strategy in which a single dose of carrier was followed by a single dose of adjuvanted subunit vaccine to elicit robust immunity (immunization factor);(4) immunization of healthy North American adult volunteers (host factors).We recognize that the use of North American volunteers may not be predictive of outcomes expected after oral delivery of candidate carrier vaccines in developing countries. This is possibly due to environmental enteropathy and therefore might only be considered as a “best possible case” for human immune responses when compared to target populations in endemic regions for which a given vaccine is intended. Despite the promise of this clinical trial, several other considerations should be examined in the future to improve the utility of the carrier vaccine approach. Either secretion or surface expression of the foreign antigen should be explicitly examined in optimized carrier strains to further improve antigen-specific immunity. In addition, most previous trials have only examined humoral responses against the foreign antigen, although several trials reported by T.F. Meyer and colleagues ,10 confiS. Typhi-based carriers as human vaccines. Although experimental animal models have proven essential to the initial design and pre-clinical testing of immunogenic carrier vaccines [S. Typhi-based carrier vaccines capable of eliciting robust protective efficacy against both S. Typhi and the unrelated foreign target pathogen as well. We have explored the retention of as many virulence properties of a candidate carrier vaccine as possible to elicit sufficient innate responses leading to robust adaptive immunity. However, we note that the strategies discussed here are not limited to enteric pathogens per se. These approaches can also be applied to non-pathogenic bacteria engineered as carrier vaccines; we refer the reader to other excellent reviews for a more in-depth examination of engineered non-pathogenic carrier vaccines [Here, we have attempted to highlight promising technologies and approaches that might be exploited to improve the success of vaccines ,125, it vaccines ,127. Reg


The integrin receptor protein talin plays vital roles in intracellular chemical and mechanical activities, and it is implicated in the high invasion and poor prognosis of non-small cell lung cancer (NSCLC). To better understand the mechanism underlying the function of talin in NSCLC invasion and metastasis, a few newly designed tension probe based on Förster resonance energy transfer was used for real-time observation of tension changes in A549 cells. High NSCLC cell aggressiveness was found to be accompanied with inward talin and outward glial fibrillary acidic protein (GFAP) tensions, which are closely associated with microfilament (MF) force and intracellular osmotic potential. The increased osmotic pressure resulted from the production of intracellular protein nanoparticles and the related ion influx. Furthermore, integrin activation was found to adjust the talin and GFAP tensions. Disruption of the interaction between talin and MFs blocked the mechanical source of talin, reducing both talin tension and osmotic pressure and thus inhibiting NSCLC cell invasion and migration. Consequently, our study demonstrates that talin is involved in NSCLC invasion and migration via its inward tension and that the integrin pathway is correlated closely with protein-nanoparticle-induced outward osmotic pressure. Lung cancer is the leading cause of cancer mortality worldwide in both men and women, and metastases are considered to be the major cause of lung-cancer-related deaths The cytoskeletal protein talin is widely implicated in cancer progression processes, such as cell polarization, migration, and metastasis Previous studies have demonstrated that the talin complex is a vital signaling scaffold in the transmission of chemical and mechanical signals in cellular physiological and pathological activities Accordingly, in the present study, we investigated the relationship between talin-related tension and the aggressiveness of NSCLC cells. A newly designed tension probe based on Förster resonance energy transfer (FRET) was used for real-time monitoring of talin and glial fibrillary acidic protein (GFAP) tensions in A549 cells. Thus, we were able to ascertain the direction and magnitude of talin tension and osmotic pressure and analyze the mechanism underlying NSCLC invasion and migration.2 at 37 °C.A549 cells were purchased from Shanghai Cell Bank Type Culture Collection Committee . Cells were cultured in Dulbecco's Modified Eagle's Medium  containing 10% fetal bovine serum  and antibiotics (100 U/mL streptomycin and penicillin) under humidified air containing 5% COAntibody against α-tubulin  was obtained from Proteintech Group . Antibody against talin  was purchased from Boster Bio . Fluorescein-conjugated goat anti-mouse IgG (H+L)  and fluorescein-conjugated goat anti-rabbit IgG (H+L)  were obtained from Zsgb-Bio .Ciliobrevin D, blebbistatin, and cilengitide were purchased from Aladdin . Ispinesib (SB-715992) was purchased from Beyotime Biotechnology . Cytochalasin D (Cyto D) and nocodazole (Noc) were obtained from MilliporeSima . Manganese chloride was obtained from Sinopharm .Osmotic pressure was measured as reported previously A549 cells were dissolved in radioimmunoprecipitation assay lysis buffer (Beyotime Bio) supplemented with phenylmethylsulfonyl fluoride  and a protease inhibitor cocktail (MilliporeSigma). Cells lysates were denatured by boiling in loading buffer. The extracted total proteins were separated using SDS-PAGE and transferred to nitrocellulose membranes, which were blocked by incubation in 5% nonfat milk for 1 h. The membranes were then incubated with specific antibodies overnight at 4 °C. After washing three times, the membranes were incubated with secondary antibodies for 2 h. Enhanced chemiluminescence (ECL) chromogenic substrate was used to visualize the immunoreactive protein bands, the intensities of which were quantified by densitometry . Actin or tubulin were used as the control.5) were starved overnight and seeded into the upper chamber in serum-free medium. Then, medium supplemented with 20% FBS was added to the bottom chamber. After 24 h incubation, the cells were stained with 4% paraformaldehyde and dyed with crystal violet. Typical images of invading cells were obtained for statistical analysis. Migration assays were performed in a similar manner to the invasion assays, but without pre-coating with matrigel.The upper chamber of a transwell apparatus  was pre-coated with 50 μL of matrigel solution. A549 cells  and the acceptor (eYFP). Thereafter, the FRET analysis was conducted as reported in previous works Fresh breast cancer tissue samples and adjacent normal tissues were obtained surgically from the Department of Surgical Oncology, The First Affiliated Hospital of Nanjing Medical University . All patients gave informed consent, and the study was ethically approved by the Institutional Review Board of Human Research of the Affiliated Hospital of Nanjing Medical University. The dates for survival analysis were obtained from the Kaplan-Meier plotter database. All patients were sorted into different groups and tested for significance using the log-rank test.Data are shown as mean ± SEM and verified using a Student's t test. One-way ANOVA was conducted using SPSS v.22.0  for single-factor sample comparisons, and a least significant difference test was used for comparisons between any two means. Each experiment was repeated at least three times, >10 cells were imaged, and each condition was analyzed.The integrin-binding adaptor protein talin is considered to be a regulator in tumor progression Then, we analyzed the expression of talin-1 in patients with NSCLC  depolymerizing agent Noc were used to identify the mechanical source of talin tension. Talin tension shows an obvious decrease in A549 cells treated with Cyto D or Noc, with Cyto D treatment causing a sharper reduction.Motor protein inhibitors Osmotic pressure, especially protein-nanoparticle-induced osmotic pressure, may play an important role in the tension activity related to cancer invasion and metastasis A GFAP-cpst-GFAP probe was constructed and transfected into A549 cells by a previously reported method - concentration - content is closely associated with the upregulation of protein nanoparticles . These results indicate that talin tension and protein-nanoparticle-induced osmotic pressure promote A549 cell aggressiveness in response to integrin activation.The size distribution of cytoplasmic particles was found to be a key influencing factor of osmotic pressure. As the intracellular osmotic pressure increases, the number of cytoplasmic protein particles less than 100 nm in size is significantly increased after MnClWe have demonstrated that the increase in talin tension results from inward MF force. To further investigate whether a connection between talin and actin mediates NSCLC invasion and metastasis, we designed a talin-truncated FRET probe , in which the actin binding domain is deleted, preventing actin-talin binding The talin tension increases more slowly after deletion of the actin binding domain in the short-term aggressive EGF model Fig. A. FurtheTalin/integrin signaling plays an important role in tumor progression as a convergency of chemical and mechanical signals As a cytoskeletal protein, talin only transmits tension and cannot produce force In a previous study, the talin-integrin complex was found to link the extracellular matrix and promote focal adhesion formation in tumor progression Cell osmotic pressure is considered to be another driving force in cancer invasion and migration Talin not only transmits MF force, it also participates in depolymerization of the cytoskeleton, increasing osmotic pressure, and thus to tumor invasion and metastasis (Fig. In summary, the present study has identified talin as a critical regulator in NSLCL invasion and migration. Talin transmits MF force and produces osmotic pressure to increase the intensity of osmotic pressure, which is regulated by integrin activity. Higher cell aggressiveness is accompanied by increased talin tension and osmotic pressure resulting from production of intracellular protein nanoparticles.Importantly, the use of FRET tension probes allowed us to better explore the magnitude and direction of intracellular tension in cancer cells, revealing that talin-related mechanical activity plays a regulatory role in tumor invasion. We thus suggest that talin-related inward tension and outward osmotic pressure may be potential targets for NSCLC therapy.


Are bariatric interventions associated with serum micronutrients and metabolic hormones changes in the years after intervention, and do trajectories differ between laparoscopic sleeve gastrectomy vs gastric bypass procedures?In this comparative effectiveness study of 499 patients who underwent sleeve gastrectomy and 189 patients who underwent one-anastomosis or Roux-en-Y gastric bypass, parameters associated with calcium metabolism did not differ between the groups. The extent of hemoglobin suppression was greater among patients who underwent gastric bypass compared with those who underwent laparoscopic sleeve gastrectomy, but the difference was not explained by differences in iron stores or vitamin B levels.The findings of this study suggest that micronutrient deficiencies after bariatric interventions are differentially associated with types of bariatric procedures. This comparative effectiveness study examines the association of bariatric interventions with micronutrient deficiencies and endocrine disturbances. Few studies have described the longitudinal trajectories of serum levels of micronutrients whose deficiencies are associated with serious sequelae following bariatric procedures, such as anemia, osteoporotic fractures, and neuropathies. Furthermore, previous studies comparing laparoscopic sleeve gastrectomy (LSG) vs Roux-en-Y gastric bypass (LRYGB) or one-anastomosis gastric bypass (OAGB) procedures may have been limited by selection and confounding biases.To appraise the spectrum and temporal course of micronutrient deficiencies associated with bone metabolism and erythropoiesis after LSG vs OAGB or LRYGB procedures, using the propensity score as a balancing score.This prospective, longitudinal comparative effectiveness study was conducted at a high-volume bariatric unit in Singapore from September 1, 2008, to November 30, 2017, with a cutoff date for analysis of September 2018. Patients who underwent adjustable gastric banding, biliopancreatic diversion procedures, and intragastric balloon procedures were excluded. All other patients who underwent bariatric procedures were included. Data were analyzed from September 23 to 30, 2018.Serial assessment of 13 biochemical parameters at 12 time points for up to 5 years after bariatric procedure. Inverse probability-of-treatment weights were used to obtain estimates of the mean associations of variables assessed with the bariatric surgical interventions. Longitudinal trajectories were analyzed using mixed-effects generalized linear models to apportion the temporal variation of serum micronutrients into fixed-effects and random-effects components.P = .70), serum 25-hydroxyvitamin D levels , or phosphate levels . Hemoglobin levels were a mean 0.63  g/dL higher among patients who underwent LSG compared with those who underwent OAGB or LRYGB (P < .001), despite no differences in iron concentration levels , total iron-binding capacity , or ferritin levels . Compared with patients who underwent LSG procedures, patients who underwent OAGB or LRYGB had higher folate levels  but lower serum magnesium levels  and zinc levels .A total of 688 patients were included in this study, of whom 499 underwent LSG  and 189 underwent OAGB or LRYGB . There were no differences during follow-up among patients who underwent LSG vs those who underwent OAGB or LRYGB in intact parathyroid hormone levels (mean difference, 7.05  pg/mL; These findings suggest that LSG vs OAGB or LRYGB procedures have differential associations with various micronutrient and metabolic parameters. These differences should be recognized in guidelines for postbariatric nutritional surveillance and prevention. In contrast, interventions with a malabsorptive component  are thought to be associated with more profound and focal nutritional deficits and metabolic derangements, as they entail bypassing alimentary tract segments involved in the absorption of specific dietary nutrients. For example, the duodenum and proximal jejunum serve as critical absorption sites for trace elements, such as zinc, copper, and calcium,2 and also contain free fatty-acid receptors that modulate cholecystokinin secretion and hence affect absorption of fat-soluble vitamins .12While effective at inducing substantial and durable weight loss, bariatric procedures are recognized to predispose patients to vitamin and mineral deficiencies.19 However, the longitudinal trajectories of serum micronutrient levels remain unclear because most previous studies have analyzed data at single cross-sectional points , which may not be representative of temporal fluctuations of micronutrient levels over time. Thus, the aim of this study was to delineate a clearer picture of the extent and severity of micronutrient deficiencies associated with laparoscopic sleeve gastrectomy (LSG) compared with laparoscopic Roux-en-Y gastric bypass (LRYGB) or one-anastomosis gastric bypass (OAGB) using longitudinal mixed-effects models.21 To minimize confounding and selection biases, analyses were adjusted using inverse probability-of-treatment weights (IPTWs) based on propensity scores.29Long-term micronutrient deficiencies are likely associated with the elevated incidence of osteoporotic fractures, secondary hyperparathyroidism, anemias, and other late sequelae of bariatric procedures.ISPOR) and Strengthening the Reporting of Observational Studies in Epidemiology nutritional epidemiology extension (STROBE-nut) reporting guidelines for comparative effectiveness research using nonrandomized cohort studies.Ethical approval to conduct this study was granted by the SingHealth institutional review board with waiver of informed consent because data were deidentified. The final data set used for analyses was retrieved on September 23, 2018. To ensure relevance to stakeholders, we reviewed the International Society for Pharmacoeconomics and Outcomes Research , Malay (15.0%), and Indian (7.4%) individuals.31 The abdominal cavity was insufflated with carbon dioxide and intraabdominal pressure maintained at 15 mm Hg. Dissection commenced at approximately 3 cm proximal to the pylorus; the omentum was separated from the greater curvature by dividing the branches of the gastroepiploic vessels and the short gastric vessels using a Harmonic scalpel (Ethicon Endosurgery). Special attention was paid in completely exposing the left crus of the diaphragm and ensuring complete clearance of the posterior aspect of the fundus. A 120-mm calibration tube was inserted orally and the stomach tubularized with the application of an endoscopic stapler . The disconnected stomach was removed in an endoscopic bag  via a 15-mm opening at the umbilicus. For LRYGB procedures, a 20-mL lesser curve-based gastric pouch was fashioned over a 120-mm orogastric tube, with a 100-cm biliopancreatic limb and 100-cm antecolic Roux limb with stapled gastrojejunostomy. Mesenteric defects were closed.32Patients undergoing bariatric interventions at our institution are treated by a specialized, multidisciplinary bariatric unit comprising bariatric surgeons, endocrinologists, and registered dietitians. All LSGs were performed using 5 ports placed in the upper abdomen as previously described.33 Per our institution’s standard operating practice, nutritional deficiencies identified prior to surgical procedures were orally replaced. After bariatric intervention, patients were prescribed the following supplements per our unit protocol: 1 to 2 multivitamin tablets daily, elemental calcium (1000-2000 mg per day), and vitamin D2 (50 000 IU 1 to 2 times per week). The multivitamin tablet in our study is a commercially available prenatal formulation that includes vitamin A (3000 IU), thiamine (10 mg), folic acid (1000 µg), copper (100 µg), and iron . Subsequent doses of supplements were titrated according to 6 to 12 monthly routine laboratory measurements. Supplemental oral iron formulations, vitamin B12, and folic acid were not standardized or enforced but could be ordered at the treating physician’s discretion if patients were deemed at risk of developing anemia  or if deficiencies were identified during follow-up. Ergocalciferol (vitamin D2), instead of cholecalciferol (vitamin D3), was the choice of vitamin D replacement in this study, as it was the only high-dose vitamin D formulation available at our institution. The high-dose vitamin D replacement regimen used at our institution is based on several endocrine society guidelines that have advocated aggressive vitamin D supplementation following a bariatric procedure.34 To ensure adequacy of vitamin D replacement without incurring toxic effects, 25-hydroxyvitamin D levels were closely monitored, and the dose of vitamin D2 was titrated to achieve a target 25-hydroxyvitamin D level of 30 ng/mL . Patient adherence to all supplements was assessed and reinforced at each visit.For the purpose of dichotomization, we classified procedures as being predominantly restrictive  or predominantly malabsorptive . However, in reality, it should be acknowledged that many bariatric interventions incur weight loss through a mixture of restrictive and malabsorptive mechanisms. All patients were dispensed standardized dietary advice regarding staged meal progression and macronutrient proportions  under supervision of registered dietitians, consistent with clinical practice guideline recommendations.12 , vitamin B9 , zinc, and magnesium. These assessments were performed at 12 predefined points  for up to 5 years.All laboratory tests were performed in the fasted state, and serum parameters included albumin, calcium, phosphate, intact parathyroid hormone, 25-hydroxyvitamin D, ferritin, iron, hemoglobin, total iron-binding capacity, vitamin B37 Individuals with missing outcome data in the immediate postoperative period were excluded from the imputation procedure. Propensity scores for individual patients were then computed by combining the postestimation probabilities across 50 multiply imputed data sets per Rubin rules.To estimate mean associations for the interventions and minimize selection and confounding biases, we performed a comparative effectiveness study using the propensity score as a balancing score. Variables were selected for inclusion into a penalized logistic regression model using a L1-regularized procedure based on the least absolute shrinkage and selection operator λ penalty, the optimal value of which was determined through 10-fold cross-validation to give the minimum cross-validated error. To enhance statistical power, baseline covariates included in the propensity score model as well as missing follow-up outcome data were multiply imputed (50 imputations) using multivariate chained equations with the following specifications: predictive mean matching for continuous variables (5 k–nearest neighbors), augmented logistic regression for binary variables, and ordinal logistic regression for ordered categorical variables .37 This was accomplished by incorporating a treatment-by-time interaction with an unstructured covariance and Huber-White robust variance estimators. The fixed-effects component can be interpreted to reflect the true underlying trajectory of the outcome parameter at the population level, while the random-effects terms are used to model interindividual and interoccasion variability. All mixed-effects analyses were adjusted using IPTW, and we additionally adjusted for sex in our models, as sex was not accounted for in the propensity score but could nevertheless represent a potential confounder because of physiological differences between the sexes. Comparisons at different points or between the 2 types of bariatric procedures were calculated using predictive margins or contrasts from the postestimation linear combinations of coefficients. Finally, we also repeated all analyses using the raw data , and the results of these sensitivity analyses are reported in the eAppendix in the To efficiently use all longitudinally repeated measurements of micronutrient and hematological or endocrine parameters, generalized linear mixed models were carried out to apportion the temporal variation of serum micronutrients into fixed-effects and random-effects components.t tests for continuous variables, Pearson χ2 test for categorical variables, and log-rank test for time lapse between diagnosis of diabetes to bariatric procedure. To assess whether there was residual statistical imbalance after conditioning baseline distributions on the propensity score, we repeated the comparisons with inverse probability weighted linear regressions for continuous variables, logistic regression for categorical variables, and Cox regressions time-to-event variables. All statistical analyses were performed in Stata statistical software version 16.0 (StataCorp). P values were 2-sided, and P < .05 was regarded to indicate nominal statistical significance.Unadjusted comparisons of baseline characteristics were performed using unpaired P = .73) , younger age , and lower-risk glycemic status   and 189 underwent OAGB or LRYGB . The propensity-score model exhibited excellent discrimination  and calibration (Hosmer-Lemeshow test  < .001) . AdditioP = .70) (P < .001) , despite the fact that vitamin D levels differed significantly between both groups at many of the individual follow-up points (P < .001), but not phosphate levels   . In the p points B. ComparP = .83) .P < .001)  compared with levels measured prior to bariatric intervention , total iron-binding capacity , or ferritin levels   g/dL  higher among patients who underwent LSG compared with those who underwent OAGB or LRYGB ( < .001) A. In botP = .71) .P < .001 for monotonicity) but not men (P = .78 for monotonicity) during follow-up . Additional results concerning the differential associations of OAGB or LRYGB and LSG with iron-related parameters in both sexes are detailed in the eAppendix, eFigure 6, and eFigure 8 in the Interestingly, in stratified analyses by sex, we detected a strongly monotonic, decreasing trend in hemoglobin levels among women (9) levels compared with patients who underwent LSG   (P < .001) and zinc  during follow-up  , which lP = .06) . Howeverollow-up .P = .09). Likewise, we also compared percentage of total weight lost between LSG vs OAGB or LRYGB groups and found no difference in percentage of total weight lost during follow-up   data without propensity score weighting, are reported in the eAppendix in the 19 and the first to use treatment propensity scores and longitudinal mixed models to address selection and confounding biases and the time-varying nutritional changes associated with predominantly restrictive  vs malabsorptive  bariatric procedures. The findings from this study can be regarded to reflect an audit of and to be generalizable to the contemporary real-world practice of many bariatric surgical units. In recent years, LSG has rapidly gained popularity to become the most frequently performed bariatric procedure, as is the case in our institution.38To our knowledge, this prospective comparative effectiveness study of postbariatric procedure nutritional outcomes is the largest from Asia, among few to comprehensively interrogate the longitudinal trajectories of micronutrient levels of which deficiencies are associated with development of late metabolic complications,2 and elemental calcium supplements were stipulated as part of our postbariatric treatment protocol, serum 25-hydroxyvitamin D levels were unsurprisingly higher at all points compared with baseline, and there was no statistically significant difference between patients who underwent LSG compared with those who underwent OAGB or LRYGB during follow-up. Interestingly, intact parathyroid hormone levels were fairly stable in our study and stand in contrast with previous findings by Johnson and colleagues,12 who reported progressively increasing parathyroid hormone levels over time. An important difference is that many historical studies used a far lower dose of vitamin D . Therefore, we surmise that the aggressive vitamin D replacement regimen used in our study is safe yet may be more efficacious than previous studies using lower dosages for preventing increases in parathyroid hormone levels.Since vitamin D39 Indeed, in our study, postbariatric procedure hemoglobin levels in the overall cohort were significantly attenuated at all points and never recovered to prebariatric procedure levels. However, patients who underwent OAGB or LRYGB recorded a steeper decrease in hemoglobin during the first 3 months compared with those undergoing LSG, and hemoglobin levels remained statistically lower in the OAGB or LRYGB group than the LSG group for the rest of follow-up. These temporal trends indicate that surveillance and prophylactic efforts against anemia should commence earlier for patients undergoing OAGB or LRYGB than those undergoing LSG, preferably within the first 3 months or even before the procedure.41Anemia is the most prevalent nutritional complication of bariatric procedures reported in the literature, occurring in as many as two-thirds of patients.This study also represents one of the first reports to delineate the interactions among sex, follow-up time, and nutritional outcomes after bariatric intervention. A novel finding is that among women, hemoglobin levels continued to decline in a strongly monotonic fashion during follow-up, whereas such a monotonically decreasing trend was not observed among men, thus indicating that nutritional support for women who underwent bariatric procedures was inadequate compared with support for men. Furthermore, hemoglobin levels were similar despite ferritin levels being consistently higher among women who underwent OAGB or LRYGB compared with LSG. This observation may reflect an unsuccessful attempt by the treating physicians to counteract the declining hemoglobin levels by prescribing iron supplements more aggressively in women undergoing OAGB or LRYGB. Recognition of such sex- and time-dependent associations of hematological complications after bariatric procedures has been largely missing from current guidelines and represents an area that warrants further investigation.12 deficiencies, it can also manifest owing to insufficient intake of zinc, copper, and vitamins A and E.19 Intriguingly, although hemoglobin levels were lower in the OAGB or LRYGB group than the LSG group, this cannot be attributed to inadequate iron intake, as serum iron, total iron-binding capacity, and ferritin concentrations were comparable between groups. In fact, folate  levels were higher in the OAGB or LRYGB group than the LSG group for most of follow-up; thus, vitamin B deficiencies are unlikely to be the cause of reduced hemoglobin synthesis in our study. A 2014 meta-analysis found that malabsorptive bariatric procedures  were associated with greater odds of vitamin B12 deficiency than LSG, but we found no statistically significant difference in serum vitamin B12 concentrations between groups.11 A possible explanation for this observation is that treating physicians may have provided more intensive prescriptions to patients who underwent OAGB or LRYGB in an attempt to remedy the declining hemoglobin concentrations in among these patients compared with those who underwent LSG. Therefore, we surmise that the comparatively lower hemoglobin levels among patients who underwent OAGB or LRYGB could potentially be associated with their lower serum zinc concentrations or perhaps deficiencies in other micronutrients, such as copper and vitamins A and E, that were not collected in our study.The etiological origin of postbariatric procedure anemia is complex and multifactorial: although anemia most commonly results from iron, folate, or vitamin B19 yet no consensus exists regarding the optimal schedule and overall length of postbariatric procedure biochemical monitoring. Furthermore, recent surveys and reviews indicate that there is significant discordance between clinical practice guidelines published by different societies regarding vitamin and trace element supplementation, and poor adherence among surgeons to any of these guidelines.44 Experts have articulated that the challenge with developing a set of universally accepted guidelines lies in the methodological aspects of studies performed in this field. First, the current evidence base concerning postbariatric procedure micronutrient deficiencies is largely predicated on studies that reported prevalence of micronutrient deficits and endocrine derangements at single time points. Unfortunately, single-point measurements may be unrepresentative because of the temporal fluctuations in micronutrient levels. Such temporal patterns are important to capture, as they may provide clinically pertinent insights into the rate at which reserves are depleted or replenished or reflect important pathophysiological nuances . Second, also stemming from the issue of single-point sampling, is the difficulty of synthesizing evidence in systematic reviews and meta-analyses if sampling schedules differ across studies. Third, many studies also convey the extent of nutritional deficits in terms of prevalence, which is problematic because the dichotomization of continuous parameters is associated with reduced statistical power and also inconsistencies in laboratory cutoff values for defining deficiencies.Micronutrient deficiencies after bariatric procedures are clinically significant,This study has some strengths. We used IPTW after fitting a propensity score model to obtain marginal contrasts of the treatment effect, which is defined as the difference in population effects if all patients in the study had been assigned to OAGB or LRYGB or to LSG. Inverse probability-of-treatment weighting is a popular method in the statistical toolbox for causal inference, as it addresses the problem of unobserved counterfactuals: since the same patient cannot possibly be assigned to both types of bariatric procedures at once, the outcome of either treatment would always be unobserved. Using IPTW overcomes this problem by creating a pseudopopulation to estimate the difference between 2 counterfactuals and estimating the treatment effects had all participants who underwent OAGB or LRYGB been assigned to LSG instead. Another analytical strength of our study is the application of generalized linear mixed-models to efficiently use all data in this longitudinal study, which allowed us to scrutinize and account for the temporal changes in serum micronutrient levels and other metabolic parameters, in contrast with many previous studies that analyzed these outcomes at single (or fewer) cross-sectional time points.30 This is notable because of the rapidly increasing popularity of bariatric procedures in Asia. The Asian bariatric procedure landscape has changed considerably as the epicenter of obesity and diabetes has shifted to Asia in recent years. Two of the world’s most populous and rapidly urbanizing countries, India and China, recorded the highest numbers of obese children in 2015, which could foreshadow a considerable future burden of overweight and obesity in these regions.50 Also noteworthy of this study is that it reports a larger and more comprehensive catalog of parameters than many previous studies, and especially focuses on calcium, iron, and vitamin B metabolism, as deficiencies of these micronutrients mediate some of the most prevalent metabolic complications after bariatric procedures .Other strengths of our study worth highlighting include this study being the largest Asian cohort to our knowledge to date, with representation by 3 major Asian ethnicities .29Our study also has some limitations, including the multiplicity of statistical comparisons, which is related to the large panel of biochemical parameters assessed at multiple points for up to 5 years of follow-up. An additional limitation is the nonrandomized nature of our data, which carries caveats, such as patient nonadherence, self-medication, and loss to follow-up. Nevertheless, the prospective nature of our data can also be regarded to represent the real-world contemporary experience of most bariatric procedure units. Furthermore, we also strived to minimize selection and confounding biases using IPTW.2 supplementation and physician-directed dose modification to mitigate some of the nutritional and metabolic disturbances associated with bariatric interventions, such as increasing parathyroid hormone levels. Future research directions include examining how prebariatric procedure energy-restricted diets could entrench micronutrient deficiencies,41 scrutinizing whether race/ethnicity and cultural factors influence micronutrition, and more detailed longitudinal phenotyping of patients using biomarkers, such as bone mineral density and osteoclast activity.The findings of this study suggest that choice of bariatric procedure may be associated with differences in trajectories of micronutrient levels related to erythropoiesis and bone metabolism. Our study also demonstrates that micronutrient levels after bariatric procedure are not constant, but rather, fluctuate over time, and this has implications for the interpretation of previous investigations that used a single-point measurement and the design of future studies in this developing research area. Furthermore, the findings of our subgroup analyses by sex suggest that guidelines for postbariatric procedure biochemical monitoring and nutritional management should recognize that women may be more susceptible to anemia and are likely to require more intense surveillance and prophylactic supplementation. Our results also support the utility of high-dose vitamin D


Desmoplastic small round cell tumor represents an unusual variety of tumors that affects mostly children and young males. In this report we present a case of a desmoplastic small round cell tumor involving the parotid gland of a young male. The tumor was excised, and definitive histology showed features of desmoplastic small round cell tumor confirming the diagnosis. Desmoplastic small round cell tumor (DSRCT) represents an unusual variety of tumors that affects mostly children and young males. The peak age of incidence is around the third decade of life and primary locations are usually soft tissues of the abdomen or pelvis, like retroperitoneum, omentum or mesenteries . DSRCT cAn 18-year-old man without medical background noticed a non-painful mass in the left parotid gland, with no other symptoms, for less than a month. No history of weight loss, fever or night sweats was reported. In the physical examination, a mobile, non-tender oval mass with a 1.5 cm diameter approximately on the left parotid gland, was found. There was no facial nerve dysfunction.Ultrasound (US) examination demonstrated a solid, polylobed and poorly-defined contour mass, about 21 mm in diameter, with small hyperechogenic focus inside, located in left parotid gland. T1-weighted magnetic resonance imaging showed a low-density and poorly-defined mass, involving both parotid lobes without extra-parotid extension, with high-intensity in T2-weighted sequences and low apparent diffusion coefficient (ADC) score (0.8)  as neoadjuvant chemotherapy, following which radical surgery and adjuvant chemoradiotherapy were recommended after discussions in the head and neck cancer committee. A left radical parotidectomy, due to gross facial nerve involvement evidenced during surgery with cervical lymph nodes dissection of level II and III, was performed. Facial nerve reconstruction was performed at the same sitting, and a free gracilis muscle flap was transferred by splitting the obturator nerve to perform two coaptation to both free ends of the facial nerve  and directly to the masseteric nerve Figure . FinallyA good initial response to treatment was observed, including a good functional outcome of the facial nerve reconstruction (House-Brackman 3/6). The patient received adjuvant chemoradiotherapy with a good response. After 14 months of follow-up, a retropharyngeal lymph node recurrence was observed and confirmed by histology. Actually, the patient has underwent a divergent cycle of chemotherapy.Desmoplastic small round cell tumor represents an unusual variety of tumors that affect mostly children and young males. The peak age of incidence is around the third decade of life and primary locations are usually soft tissues of the abdomen or pelvis, like retroperitoneum, omentum or mesenteries . Extra-aDSRCT cases in major salivary glands have been reported but in a very small number of patients. The most common mesenchymal salivary gland tumor described across the literature is the lipoma, but malignant histology is uncommon . About dPrimary small cell undifferentiated carcinoma of the salivary glands represents around 2% of all salivary gland tumors and are more common in older patients than the DSRCT. Molecular biology studies are very useful in the differentiation between these tumors . PrimaryThe histology of this neoplasm is very characteristic, usually composed of nests of small round blue cells with a prominent desmoplastic, fibromyxoid or collagenous stroma . The immHowever, DSRCT has a poor overall survival despite initially possible good response to a multimodal treatment protocol, including surgery, chemotherapy and radiotherapy . To the Although DSRCT in the major salivary glands is extremely rare, it should be included in the differential diagnosis of poorly differentiated salivary gland tumors, especially in adolescent and young adult males. Survival is low despite their initial response to multimodal treatment. Most patients relapse with disseminated disease that is unresponsive to further therapy.


Following their introduction a decade ago, electronic cigarettes (e-cigarettes) have grown in popularity. Given their novelty, knowledge of the health consequences of e-cigarette use remains limited. Epidemiologic studies have not comprehensively explored associations between e-cigarette use and hypertension, a highly prevalent health condition and major contributor to cardiovascular disease burden. In this study, cross-sectional associations of cigarette smoking and e-cigarette use (vaping) with self-reported diagnosed hypertension were evaluated among 19,147 18–55 year old respondents in Wave 3 (2015–2016) of the Population Assessment of Tobacco and Health Study. Multivariable analyses first modeled smoking and vaping as separate 2-category variables, then as a 6-category composite variable accounting for former smoking. After adjusting for potential confounders, current vaping  and current smoking  were both associated with higher odds of hypertension. In analyses modeling smoking and vaping compositely, respondents who were concurrently smoking and vaping had the highest odds of hypertension . These results differ somewhat from prior epidemiologic studies of vaping and respiratory outcomes, which consistently report smaller point estimates for current vaping than for current smoking. Our findings reinforce the uncertainty surrounding long-term health consequences of vaping, as well as highlight important distinctions between respiratory and cardiovascular outcomes when considering the harm reduction potential of e-cigarettes. Tobacco’s status as a leading cause of preventable disease and premature mortality spans many decades . While iIn light of the detriment caused by smoking tobacco, electronic cigarettes (e-cigarettes) were developed as alternatives to combusted cigarettes in the late 2000s. E-cigarettes encompass a range of devices which heat and aerosolize a solution that typically contains nicotine and a mixture of propylene glycol, glycerin and various flavoring additives. Laboratory studies have shown that the amount and concentration of toxicants in e-cigarette aerosol are substantially lower than in cigarette smoke ,8,9. As The potential public health implications of smokers fully transitioning away from cigarettes in favor of vaping are still not well understood. Knowledge regarding associations between vaping and a multitude of clinical health outcomes, including cardiovascular conditions, is currently limited. A handful of published studies have examined cross-sectional associations between CVD and vaping in large free-living samples of the US adult population ,12,13,14Notably, epidemiological studies have yet to examine associations between vaping and important clinical risk factors of CVD , most of which are known to be adversely associated with smoking cigarettes. Of particular interest is hypertension, as it remains the leading risk factor for CVD worldwide, and for which an estimated 10.4 million deaths and 218 million disability-adjusted life years were attributed in 2017 . TransieGiven the equivocal state of evidence for cigarette smoking and a lack of evidence for vaping, the present study evaluated cross-sectional associations of vaping and cigarette smoking with self-reported hypertension in a nationally representative sample of US adults, with a focus on young and middle-aged adults. Multiple statistical modeling approaches were employed in an attempt to scrutinize the association between vaping and hypertension independently from cigarette smoking, as well as approximating cumulative exposure to both products together.https://www.icpsr.umich.edu/web/NAHDAP/studies/36498/datadocumentation (accessed on 8 March 2021)). The PATH Study is a nationally representative prospective cohort study evaluating tobacco use behaviors, perceptions, and tobacco-related health outcomes among youth and adults in the United States ) current vapers and 5654 (19.5% [18.7–20.3]) current smokers . Most cuOverall, 17.3% (16.4–18.1) of respondents had self-reported hypertension in 2015–2016 . Self-reMultivariable results from modeling smoking and vaping as a composite variable are shown in In this nationally representative cross-sectional study of US adults aged 18–54 years, both current vaping and current smoking were significantly associated with self-reported diagnosed hypertension when modeled as separate parameters and controlling for potential confounding variables. The strength of association was similarly modest for both factors: current vapers had 31% higher odds of hypertension than those who did not currently vape, while current smokers had 27% higher odds of hypertension than those who did not currently smoke. Additionally, when modeling a combined smoking and vaping variable that accounted for former smoking, current dual users had 77% higher odds of hypertension, while current exclusive smokers had 36% higher odds. Although the small number of vapers in the analytic sample limited statistical power upon additional stratification, the association between current exclusive vaping and hypertension appeared slightly stronger among those who formerly smoked.This was the first epidemiologic study to comprehensively evaluate the association between vaping and hypertension in an adult sample. Vaping is most common among three distinct groups of individuals: (a) youth and young adults, many of whom have never been habitual cigarette smokers, (b) adult current smokers who are interested in quitting, and (c) adult former smokers who have successfully quit smoking but continue vaping in place of cigarettes ,25. WherEven with these limitations in mind, our findings are concerning from a public health perspective. Hypertension is a relatively common condition  and playGiven the recency of e-cigarettes, the long-term health effects of vaping remain unclear. Thus far, evidence supporting a hypothetical role for vaping in hypertension development primarily stems from animal models. A handful of mechanisms have been established in studies of mice regularly exposed to high levels of e-cigarette emissions. These include overactivation of the renin–angiotensin–aldosterone system via vaping-induced increases in circulating levels of inflammatory cytokines  and heigWhile knowledge of the absolute harms of e-cigarettes remains limited, there have been some randomized controlled trials (RCTs) that provide valuable insight towards the relative harms of vaping compared to smoking, including two that explored blood pressure as a secondary outcome. The ECLAT study was a 1-year smoking cessation trial where adult smokers were given one of three e-cigarettes of varying nicotine concentrations with the aim of quitting smoking. Among respondents who fully abstained from smoking from week 12 to the end of follow up (n = 18), no significant changes in systolic blood pressure were observed for baseline normotensive participants (systolic blood pressure < 130 mmHg and diastolic blood pressure < 85 mmHg), while those with baseline elevated blood pressure saw an average 16.3 (standard deviation: ±11.3) mmHg reduction in systolic blood pressure . In the In addition to the aforementioned RCTs which have considered blood pressure as a secondary outcome, Polosa et al. explored blood pressure changes in a 2016 prospective analysis of 89 baseline hypertensive smokers  . 20 of tIn addition to being the first epidemiologic study of vaping and hypertension, our study has a handful of strengths. The PATH Study is an exceptionally comprehensive data source with respect to tobacco use, even including biomarker data for a large subset of participants. This affords a unique opportunity to validate self-reported tobacco use measures that other epidemiologic datasets may not have. Indeed, prior assessments of specificity and sensitivity for self-reported smoking and vaping status instill confidence that exposure misclassification is not a major concern in our analysis . PATH’s 2p <0.001), however the absolute differences in proportions between groups were for not especially large. Finally, the e-cigarette marketplace has evolved since the time of this survey to include devices of varying power as well as substantial ranges in nicotine concentration [Along with previously mentioned limitations, a constraint of PATH is the lack of dietary information, which is particularly relevant for hypertension  . As healntration ,57. It wIn summary, this cross-sectional analysis of young and middle-age adults in Wave 3 of the PATH Study found a positive albeit weak association between vaping and self-reported hypertension, of similar magnitude to that of cigarette smoking and hypertension. Our findings underscore the importance of more rigorous longitudinal research into the health effects of e-cigarettes, reinforcing the uncertainty surrounding long-term ramifications of vaping. Moreover, the results suggest important distinctions should be made between respiratory and cardiovascular outcomes when considering the harm reduction potential of e-cigarettes.