Background. There is a high incidence of inconclusive cytopathology at initial EUS-FNA (endoscopic ultrasound-guided fine-needle aspiration) for suspected malignant pancreatic lesions. To obtain appropriate preoperative or palliative chemotherapy for pancreatic cancer, definitive cytopathology is often required. The utility of repeat EUS-FNA is not well established. Methods. A retrospective cohort study was conducted evaluating the yield of repeat EUS-FNA in determining a cytological diagnosis in patients who had undergone a prior EUS-FNA for diagnosis of suspected malignant pancreatic lesions with inconclusive cytopathology. The wait times to the second procedure and to decisions regarding therapy were calculated. Results. Overall, 45 repeat EUS-FNA procedures were performed over seven years for suspected malignant pancreatic lesions. Cytopathological class (I to IV) changed between first and second EUS-FNA in 32 patients (71%). Of 34 patients with an initially nonconclusive diagnosis, 20 had a conclusive diagnosis (59%) on repeat EUS-FNA. The cumulative yield after repeat EUS-FNA for definite pancreatic adenocarcinoma was 7 (16%). The median time interval between first and second EUS-FNA was 31 (7–175) days. Conclusions. A substantial number of patients had a definitive diagnosis of adenocarcinoma on repeat FNA and were, therefore, subsequently able to access appropriate care.  Pancreatic adenocarcinoma carries a poor prognosis, with a 5-year survival rate of less than 10% . To guidEUS has become vital in the diagnosis of pancreatic cancer. Studies have demonstrated that EUS is more sensitive than both transabdominal US and CT scan in detecting solid pancreatic masses, and EUS-FNA has emerged as an essential diagnostic tool for pancreatic cancer , 5. A reDespite the demonstrated diagnostic sensitivity of EUS-FNA, there is a relatively high rate of inconclusive diagnoses resulting from the initial EUS-FNA, at 6%–11% , 11. An In many Canadian jurisdictions, access to appropriate therapy for initial management of pancreatic cancer requires a cytopathological diagnosis of pancreatic adenocarcinoma (class IV or V cytology on FNA as per Papanicolaou class ). It is,Although there are some studies evaluating a second biopsy in the setting of pancreatic lesions, the yield of repeat EUS-FNA and its effect in terms of delay to treatment or treatments offered has not been studied. This study aims to report the outcomes of repeat EUS-FNA for Canadian patients with suspected pancreatic cancer and to evaluate how access to therapies for pancreatic cancer is influenced by a repeat procedure. Results. All patients included in the study underwent repeat EUS-FNA for investigation of a pancreatic mass suspicious for malignancy, after initial EUS-FNA results demonstrated inconclusive pathology . Four patients with likely pancreatic adenocarcinoma (class IV pathology) on first EUS-FNA underwent repeat EUS-FNA, the reasons for which are described further in Results. Sequential patients who had undergone a repeat procedure for this indication during the study period were included in the study. All patients provided written informed consent to undergo both the initial and the repeat procedures. This study was approved by an Institutional Review Board at the University of British Columbia.This retrospective cohort study was conducted at St. Paul's Hospital in Vancouver, British Columbia. St. Paul's Hospital is a tertiary referral center in downtown Vancouver. Two trained and experienced therapeutic endoscopists in the gastroenterology department perform EUS-FNA. All patients in this study were selected from a database of patients who had undergone repeat EUS-FNA for investigation of a pancreatic mass between 2007 and 2014. The total number of individual patients undergoing EUS for all indications (not exclusively for investigations of pancreatic masses) at St. Paul's Hospital over the study period was 3035. These patients were identified from billing codes. The total number of individual patients undergoing a second EUS for any indication was 182 over the study period. The total number of patients undergoing a second EUS-FNA for investigation of a pancreatic mass over the study period was 45. The indication for EUS-FNA was determined from clinic notes. Clinical presentation and location of lesions leading to the first EUS-FNA are described further inClinical data was obtained from patients' operative notes, clinical notes, and referral letters. Pathological data was obtained from pathology reports for each patient—cytology was obtained from final pathological reports reported by attending pathologists at St. Paul's Hospital. Cytology was coded according to pathological class as follows: indeterminate (class I), negative for malignancy (class II), atypical cells (class III), likely pancreatic adenocarcinoma (class IV), diagnostic for pancreatic adenocarcinoma (class V), neuroendocrine tumor (NET), or lymphoma . Data re Results). Doppler sonography was used to identify vascular structures. The number of passes was not specifically recorded in this study and varied among patients. In general, multiple passes were made . FNA technique varied between gastroenterologists. Generally, the needle was inserted into the target lesion with the stylet inserted. The stylet was then removed and to-and-fro motions were made with or without suction, or the stylet was extracted slowly while to-and-fro motions were made within the lesion. Over the study period, the method for cytopathological preparation changed from placement of the specimen on air-drying slides with subsequent preparation and analysis by on-site cytology technologist to placement of the specimen directly into CytoLyt®  fixative. The specimens were then transported to the hospital laboratory where they were prepared and evaluated by a pathologist.All procedures were performed by two trained and experienced therapeutic gastroenterologists. EUS-FNA was performed using a linear echoendoscope, with various needles and calibers (reported inn (%) unless otherwise specified. Data analysis was conducted using statistical software . Fisher's exact test was used to compare categorical variables. Student's t-test was used to compare continuous variables. P values were calculated as 2-tailed and a value of ≤0.05 was interpreted as significant.Continuous variables were reported as median (range) and discrete variables were expressed as n = 28). Symptoms reported at initial presentation included abdominal pain , jaundice , and weight loss . In 12 patients, pancreatic cancer was suspected on the basis of incidental imaging findings (27%). The location of the mass was the pancreatic head/uncinate in 34 patients (75%), pancreatic tail in seven (16%), and pancreatic body in four (9%). The mean lesion size in greatest axis was 2.6 cm (±1.3 cm).Forty-five individual patients underwent repeat EUS-FNA of a suspected pancreatic adenocarcinoma during the study period. The mean patient age was 67.3 years (±9.5 years), and the majority of study subjects were male  changed between first and second FNA in 32 patients (71%) . Seven pThere were no definite complications associated with a repeat EUS-FNA. There were two emergency room presentations in patients with a recent repeat EUS-FNA: one was abdominal pain of unknown etiology in a patient presenting the emergency department five days after repeat EUS-FNA; the second was a nonspecific and unrelated presentation to the emergency department one day after repeat EUS-FNA. Neither presentation to the emergency department resulted in a hospital admission or a discernable change in management.P = 0.73). Nine patients without evidence of adenocarcinoma on repeat biopsy were offered surgery. This was because of a third EUS-FNA demonstrating adenocarcinoma (1), a high degree of suspicion for cancer but technically difficult EUS-FNA leading to laparotomy diagnostic for adenocarcinoma (1), ongoing suspicion for malignancy due to progression of mass on imaging with worsening symptoms (5), and the presence of a NET on second EUS-FNA (2). Chemotherapy was offered to three patients without evidence of adenocarcinoma on second EUS-FNA. This was because of enlargement and mass effect of a lymph node felt to be metastatic pancreatic cancer (1), a third EUS-FNA diagnostic of malignancy (1), and enlargement of pancreatic mass on imaging, plus ongoing symptoms in patient (1). Ten of 35 patients (29%) with indeterminate, negative, or atypical pathology underwent a third EUS-FNA at our site during the follow-up period. At one year, among patients with a likely or definitive diagnosis of pancreatic adenocarcinoma on repeat FNA (class IV or V), 6 of 10 (60%) had survived, compared with 27 of 35 (77%) patients with a repeat FNA that was not diagnostic of pancreatic adenocarcinoma (P = 0.42). Two-year survival data was available for 42 of 45 patients (three patients with recruitment from May 2013 to May 2014 and therefore incomplete two-year follow-up). At two years, among patients with a likely or definitive diagnosis of pancreatic adenocarcinoma on repeat FNA (class IV or V), 1 of 9 had survived (11%), compared with 19 of 33 (58%) patients with a repeat FNA that was not diagnostic of pancreatic adenocarcinoma (P = 0.02)  .Additional information was gathered regarding the ultimate diagnoses for patients with indeterminate (class I), negative (class II), or atypical (class III) pathology on second EUS-FNA.Of seven patients with a second EUS-FNA demonstrating indeterminate (class I) pathology, there was detailed follow-up information available for seven patients (100%). One patient underwent an open biopsy demonstrating diffuse large B-cell lymphoma. Two patients underwent third EUS-FNA that were again indeterminate; masses in both cases were ultimately thought to be benign and cystic. One patient developed gastric outlet obstruction as a result of the pancreatic mass and died from complications of this; a tissue diagnosis was never made. Two patients had third EUS-FNA demonstrating likely (class IV) pancreatic cancer. One patient had repeat EUS-FNA again indeterminate; however PET scan demonstrated metastatic disease and patient died before a tissue diagnosis was made.Of 16 patients with a second EUS-FNA demonstrating negative (class II) pathology, there was detailed follow-up information available for ten patients (62%). Four patients were ultimately diagnosed with pancreatic cancer. One patient had an open biopsy and was diagnosed with metastatic renal cell carcinoma. Two patients had repeated EUS-FNA that were again negative and did not receive clear diagnoses at last follow-up. Two patients were diagnosed with pancreatitis. One patient was diagnosed with a pancreatic cyst. Of the patients with a second EUS-FNA that was negative (class II) and who subsequently died, information on cause of death was available for four patients, and in all four patients, cause of death was attributed to complications of pancreatic cancer. In one of these four patients, diagnosis of pancreatic cancer was made on repeated EUS-FNA; the remainder were diagnosed by imaging and clinical suspicion. One of these patients underwent surgical resection, which demonstrated pancreatic adenocarcinoma.Of eight patients with a second EUS-FNA demonstrating atypical (class III) pathology, there was detailed follow-up information available for six patients (75%). One underwent Whipple resection and was diagnosed with metastatic RCC. One patient underwent repeated EUS-FNA demonstrating negative (class II) pathology and one was with likely (class IV) pathology. One patient had an open biopsy demonstrating NET. Three patients were ultimately diagnosed with pancreatic adenocarcinoma: one by repeated EUS-FNA and two based on imaging findings and clinical suspicion.Studies have consistently demonstrated the diagnostic utility of repeat EUS-FNA in patients where a diagnosis of pancreatic cancer is strongly suspected, but initial EUS-FNA is negative or nondiagnostic –18. ThisThe primary aim of this study was to explore how repeat FNA facilitated access to treatment for pancreatic adenocarcinoma. Repeat EUS-FNA is often used in a specific patient population where the pretest likelihood of cancer is high enough to warrant a second diagnostic procedure, but not high enough to proceed directly to surgery . PatientTreatment was offered to relatively few patients in the total patient group and was offered at similar rates between those patients that had adenocarcinoma likely or diagnosed on repeat FNA (class IV or V) and those who did not. The low observed rates of offered treatment in this population are in keeping with the general rates of treatment in newly diagnosed pancreatic cancer and likely due to the cancer stage at diagnosis or the poor functional status of these patients . StagingIf the goal of repeat EUS-FNA is to facilitate access to care, one would hope to observe more treatments offered to those with a definitive cytopathological diagnosis made on repeat FNA than to patients with negative or inconclusive results on repeat EUS-FNA. Patients in this study waited a median of 31 days for a repeat procedure with the goal of achieving definitive cytopathological diagnosis and were not offered surgery or chemotherapy at a significantly different rate than those without cytopathology suggestive of cancer. Additionally, a repeat diagnostic procedure is not without risk and can be associated with complications that in this study population were minor, but in other larger studies have been important . Of noteOther limitations of this study include the small sample size and the relatively few number of patients in the group with a diagnosis of adenocarcinoma (class IV or V pathology). Different pathologists reviewed initial and repeat pathology, and there may be interreviewer variability in the interpretation of cytopathology. Given the small number of patients in this study, this is an important limitation of these results. Also, this data is from a single center, and it is possible that varying practices exist elsewhere with respect to offering treatment to patients with suspected pancreatic cancer in the absence of definite cytopathology. Additionally, since this was a single-center study, there were likely patients who underwent a first EUS-FNA at our center with inconclusive pathology and who went on to have a second EUS-FNA at a different center and therefore follow-up details were not included in this study. This decreases the overall number of patients in the present study and may bias the study sample to those that were well enough to travel to our site for their second EUS-FNA or be seen in follow-up. Unfortunately, few of the study patients ultimately underwent surgical resection and therefore do not have final surgical pancreatic pathology. As such, we cannot comment on the sensitivity and specificity of repeat EUS-FNA in this population. Further investigation in this study population is warranted to directly compare meaningful clinical endpoints and treatment rates between patients with an uncertain initial FNA who do and do not undergo a repeat EUS-FNA.In conclusion, repeat EUS-FNA can alter the pathologic diagnosis and lead to a definitive diagnosis in many patients with pancreatic adenocarcinoma. In this patient population, however, this did not lead to a significant increase in the rate of treatment that was offered or administered and was associated with a substantial delay in time between first and second EUS-FNA.


Plasmodium spp., and of designing vaccines against chosen antigens found on the parasite's surface. Given the extraordinary resources exhibited by Plasmodium to escape these traditional strategies, which have not been able to free humankind from the scourge of malaria despite much effort invested in them, new concepts have to be explored in order to advance toward eradication of the disease. In this context, amyloid-forming proteins and peptides found in the proteome of the pathogen should perhaps cease being regarded as mere anomalous molecules. Their likely functionality in the pathophysiology of Plasmodium calls for attention being paid to them as a possible Achilles’ heel of malaria. Here we will give an overview of Plasmodium-encoded amyloid-forming polypeptides as potential therapeutic targets and toxic elements, particularly in relation to cerebral malaria and the blood–brain barrier function. We will also discuss the recent finding that the genome of the parasite contains an astonishingly high proportion of prionogenic domains.The main therapeutic and prophylactic tools against malaria have been locked for more than a century in the classical approaches of using drugs targeting metabolic processes of the causing agent, the protist  Anopheles mosquito inoculates during a blood meal sporozoites of the malaria parasite, the apicomplexan protist Plasmodium spp., which migrate through the skin into the circulation and then to the liver. In a few minutes, sporozoites invade hepatocytes, where they will transform into merozoites that enter the circulation to invade red blood cells (RBCs) [Malaria infection starts when a parasitized female s (RBCs) . Merozois (RBCs) . Even ths (RBCs)  which lePlasmodium species are particularly high, especially in the proteome of the Plasmodium falciparum sporozoite. Several Plasmodium IUPs have been examined as potential targets of interest for protective immune responses because repeats present in many of them are highly immunogenic showing strong reactivity with antibodies. However, antibodies raised against these repeats do not mediate protection in front of the parasite, probably because IUPs adopt more ordered structures when interacting with different ligands, and antibodies induced by such regions may recognize a variety of antigen conformers and thus react poorly with the antigen on the parasite surface. The structural plasticity of IUPs, which allows promiscuous binding interactions, may favor parasite survival both by inhibiting the generation of effective high affinity antibody responses and by facilitating the interactions with host molecules necessary for attachment to the vascular endothelium and for the invasion of host cells. Amyloidogenesis is undergone by a diverse group of evolutionarily unrelated proteins from different organisms, all sharing permanent or transiently disordered structural regions with propensity to form β-sheet aggregates in their complete or fragmented forms.Sequence analysis has shown that apicomplexan parasite proteomes are enriched in intrinsically unstructured proteins (IUPs) , which cP. falciparum, merozoite surface protein 2 (MSP2) is unusually hydrophilic and contains tandem sequence repeats characteristic of IUPs [P. falciparum is high, and the MSP2 isoforms 3D7 and FC27 have been shown to elicit significant antibody responses in clinical studies [in vitro [in vitro into fibrils with amyloid-like properties [Among several merozoite surface proteins being assessed as potential components of a vaccine against  of IUPs . MSP2 is studies . Recombiin vitro . The foroperties   filaments anchored at their bases to the plasma membrane, as in other species of Plasmodium, such as P. knowlesi [Amyloid-like protein aggregates are associated with the surface of several microorganisms . Functioknowlesi  , we can possibly conclude that both MSP2 signal peptides are at least as amyloidogenic as the amyloid β peptide (Aβ). The N-terminus amyloidogenic region within the signal peptide is probably lost at the beginning of the protein transit through the endoplasmic reticulum (ER), whereas the C-terminus sequence within the GPI anchor signal peptide is excised later in the polypeptide processing. Because MSP2 is very abundant on the merozoite surface [Somewhat surprisingly, analysis of the peptide sequence of MSP2 isoforms 3D7 and FC27 with the amyloid sequence-predicting algorithms  FoldAmyl protein . In Tabl surface , these tin vitro assays to explore if both peptides formed amyloid-like structures in agreement with the theoretical predictions presented above. Two peptides derived from the MSP2 GPI anchor and signal sequences, SSNIASINKFVVLISATLVLSFAIFI-KKKK , and MKVIKTLSIINFFIFVTFNI-KKKK , both >95% purity according to chromatographic analysis, were incubated in phosphate buffered saline (PBS) at 37°C for 2–3 weeks. Since in both peptides the amyloidogenic region starts after or with a lysine , medium  and low immunoreactive . In each group, half of the samples were malaria positive by PCR. In addition, 11 sera from Equatorial Guinea patients with MSP1 and/or MSP9-specific IgGs were also examined. Malaria nonexposed control sera were used to determine the baseline signal; all serum samples were from blood donors that had provided informed consent and the procedure was approved by the clinical research ethical committee at the University Hospital of Getafe (Spain). The analysis of the recognition of the aggregated forms of MSP2 signal and anchor peptides by all sera did not show any values above the threshold in ELISA assays. Although one interpretation of this result is that such forms are not present in vivo, we cannot rule out the possibility that amyloidogenic peptide forms have low immunogenicity, as it has been described previously [Plasmodium might have evolved amyloid-forming regions in proteins that are at some time exposed to the plasma.As a first approximation to study the possible Plasmodium genome were related to some aspect of malaria pathophysiology, such as cerebral malaria (CM), the most severe neurological complication of falciparum malaria. Gene expression analysis suggested that CM and AD share common mechanisms of pathogenesis, as indicated by the accumulation of Aβ in brains of CM-susceptible (CM-S) but not in CM-resistant (CM-R) mice [Pathogenesis in Alzheimer's disease (AD) is linked to the accumulation of the highly amyloidogenic self-associating Aβ, whose oligomers, which are intermediate species in the assembly of fibrils, are powerful neurotoxins and might therefore be the key effectors of cytotoxicity . We deci-R) mice , in agre-R) mice . This re-R) mice . The ree-R) mice , has bee-R) mice . In addi-R) mice .Plasmodium to replicate while evading splenic clearance [Plasmodium-induced sequestration of infected and uninfected erythrocytes changes blood–brain barrier (BBB) function [Plasmodium merozoites from the pRBC, among which perhaps neurotoxic amyloids formed by MSP2 signal peptides. Amyloid structures have been described to bind glycosaminoglycans such as the chondroitin and heparan sulfates of cell-bound glycoproteins and proteoglycans, including those present on ECs of the BBB [P. falciparum E8B strain and free parasites showed significant adherence to confluent human brain microvascular endothelial cells (HBMECs) in an in vitro BBB model (Erythrocytes infected with mature stages of the malaria parasite bind to the endothelial cells in the capillaries of tissues in a phenomenon known as sequestration, which allows learance . pRBCs clearance . There ifunction . Entry ofunction . pRBC bi the BBB . ErythroBB model . AlthougPlasmodium-encoded protein unequivocally related to amyloid structures of potential pathological importance in malaria. However, recent data strongly suggest that in the short term, we will witness an important increase in the number of works dealing with the role in Plasmodium cell biology of a particular type of amyloids. When amyloid fibrils grow and divide with high efficiency they can propagate, become infective and transmit aggregative folds and are then termed prions [Plasmodium proved that they might encode abundant hydrophilic low-complexity protein fragments corresponding to species-specific, rapidly diverging regions that could form nonglobular domains helping the parasites to evade the host's immune response [P. falciparum the proportion of putative prions rises to an astonishing 10%. One in four proteins in this organism contains asparagine repeats, whose functions are still unknown [Plasmodium forces the organism through drastic changes in its environmental temperature, including cyclical fever episodes that can last several hours and exceed 40°C. Because heat shock increases the propensity of proteins to misfold and form aggregates, if only a fraction of the abundant prion-like domains in the malaria parasite aggregates during heat shock, this would certainly lead to the pathogen's demise. However, Plasmodium is able to survive with one of the most aggregation-prone proteomes in nature. This is due to the presence of specialized chaperones which prevent aggregation and are essential for parasite survival within RBCs [Plasmodium suggests that they might provide certain advantages, but the functional implications of such an amount of aggregation-prone proteins are still unclear. Identifying their so far unknown roles and mechanisms of action can provide new unexpected targets for future chemotherapeutic and prophylactic antimalarial approaches.MSP2 has been the first d prions . While pd prions . There aresponse . Analysiresponse . In most unknown , but arehin RBCs . AlthougPlasmodium proteome of amyloidogenic peptides suggests that these entities have a physiological function in the life cycle of the pathogen. Functional amyloids might have roles in the adhesion of the parasite to its host cells during invasion, or participate in some of the pathological effects of malaria related, for example, to blood–brain barrier alteration or the triggering of immune responses. However, Plasmodium-derived amyloids could also provide new prophylactic or therapeutic possibilities if we are capable of harnessing them for future innovative antimalarial strategies.The growing experimental evidence for the presence in the Plasmodium, MSP2, forms amyloid-like fibrils in vitro.At least one protein expressed by Plasmodium proteins might be released in the plasma.Amyloid-like structures formed by certain signal peptides of Cerebral malaria and amyloid-related Alzheimer's disease share common mechanisms of pathogenesis.Plasmodium-infected erythrocytes show adhesion to the blood–brain barrier according to in vitro assays.Plasmodium encodes one of the highest proportions of putative prion sequences.The genome of


When a porous material is inserted into a uniform magnetic field, spatially varying fields typically arise inside the pore space due to susceptibility contrast between the solid matrix and the surrounding fluid. As a result, direct measurement of the field variation may provide a unique opportunity to characterize the pore geometry. The sensitivity of nuclear magnetic resonance (NMR) to inhomogeneous field variations through their dephasing effects on diffusing spins is unique and powerful. Recent theoretical and experimental research sheds new light on how to utilize susceptibility-induced internal field gradients to quantitatively probe the microstructure of porous materials. This article reviews ongoing developments based on the stimulated echo-pulse sequence to extend the characterization of porous media using both spatially resolved and unresolved susceptibility-induced internal gradients that operate on a diffusing-spin ensemble. Magnetic susceptibility contrast-induced inhomogeneous magnetic fields show a fingerprint of the underlying pore geometry when porous media are subjected to an external, uniform magnetic field. Nuclear magnetic resonance (NMR) provides a unique opportunity for nondestructively detecting such internal field variations, and susceptibility contrast-based NMR methods hold great promise for investigating the structural and functional properties of porous media.For example, fluid-filled rocks, soils, or tissues , which are comprised of at least two phases with different magnetic susceptibilities, give rise to an inhomogeneous internal magnetic field across their pores when embedded inside a uniform magnetic field. The presence of a nonuniform field often influences NMR-relaxation experiments and diffusion measurements. This effect was first recognized by Brown . Since tT2*- and T2-relaxation rates. As a result, Dennies et al. reported that the size of tumor microvessels can be determined by measuring the in vivo ratio between susceptibility contrast-induced T2* and T2 in the presence of an iron-oxide intravascular contrast agent  was strongest along the z-direction (the direction of the main magnetic field), and this reduction was similarly smaller along the x- and y-directions in the gradient. The anisotropy ratio of the reduction in the experimental measurements (2.2:0.92:1) was also in good agreement with the theoretical calculations of κ(0) (1.8:0.97:1) along z, x and y directions. K = [C1(r‖) − b]/[C1(0) − b]) for different bead sizes and gradient directions are shown in q-space mapping is expected to yield a complete description of the correlation function of internal magnetic fields along arbitrary directions and resolve the residual discrepancies between theory and experiment in in vivo clinical realm [Comparison between the numerically and experimentally measured field correlation functions (al realm ,66,67, tSusceptibility contrast is ubiquitous in materials, and MR is uniquely sensitive to magnetic field variations caused by susceptibility contrast. Characterization of the internal magnetic field properties of porous media and the development of new, relevant MR pulse sequences have the potential to provide important structural and functional information regarding porous media via nondestructive techniques.in vivo trabecular bone and microvasculature imaging are in progress.This paper reviews the basic concept of DDIF contrast and its extension to imaging. Appropriate data-analysis methods for bulk- and DDIF-imaging experiments have been discussed. DDIF-imaging experiments with 2D cylindrical phantoms show the direct correlation between DDIF contrast and the strength of the local internal gradients due to susceptibility differences. Interpretation of bulk-DDIF contrast in trabecular bone samples was confirmed using both micro-imaging experiments and internal field calculations of the same samples. A new PFG-NMR method for measuring magnetic field correlations was presented, and representative experiments and theoretical calculations are described for a randomly packed system of spheres, showing the potential for utilizing the statistical features of internal fields to obtain structural information. Potential applications of this method for use in


Culex pipiens complex (Cx. pipiens quinquefasciatus in Southern USA) play a critical role in the spillover of urban arboviruses such as West Nile virus or St. Louis encephalitis virus. Field studies have shown strong correlation between the periodicity of rainfall events and larval proliferation. However, mechanistic determinants driving this relationship are poorly understood. We hypothesize that rainfall events decrease strain from intraspecific competition through the associated reduction of immature density and the introduction of detritus.Members of the . Rain events were simulated in a competition-based metabolic age model and compared to a null model including environmental variability. Variable rain delays in two-event simulations showed optimal proliferation occurring with rain delays between 16 and 21 days when including density-dependent effects.To address our hypothesis, we used laboratory competition experiments to inform a deterministic matrix projection model consisting of an age-structured larval matrix coupled with a stage-structured adult mosquito matrixCx. quinquefasciatus proliferation rates can be modeled mechanistically as a density-dependent system. The empirical understanding of density-dependence as it relates to environmental stochasticity provides a theoretical platform for the study of larval dynamics and the impact of larval control in this medically relevant disease vector.These results are comparable to the pattern observed in natural populations, indicating that The online version of this article (10.1186/s13071-018-2711-1) contains supplementary material, which is available to authorized users. Culex pipiens complex (predominantly Cx. pipiens and Cx. quinquefasciatus) have been identified as the principal vectors of flaviviruses such as West Nile virus (WNv) and St. Louis encephalitis virus (SLEv) , 3433, 3Cx. quinquefasciatus populations to rainfall periodicity, 100% flushing of eggs and pupae more closely resembles the timing of proliferation blooms observed in the field.The equilibrium distribution of Model N* was used to define initial populations of double-event simulations for both Model N* and A*. Total productivity estimated by Model N* in double-event simulations increased exponentially towards baseline proliferation of single-event simulations only after rainfall periodicity was greater than 40 days. Variable impact of rainfall on flushing of immatures  produced the same qualitative trend  that present low efficacy on larval mortality in a catch basin [During the stable population period of single-event simulations , per capita survival to pupation was higher than in the volatile, exponential growth period of colonization, suggesting increased survivorship and metabolic efficiency at lower abundance or at high nutrient levels. Knowledge of population age distribution within a system and how that system responds to nutrient loading is therefore critical to understanding population response to environmental disturbance regimes or larvicide application. Insecticide-based larvicides  recreated patterns of environmental response described in the literature, we are aware of other potential factors that can influence such population behavior. In addition to the influence of detrital loading on larval populations, natural systems are sensitive to temperature effects and rainfall intensity. Unaccounted for in the simulated system, these parameters may influence the speed and response of populations to environmental stochasticity. Future work will aim to expand our modeling effort to include more realistic environmental drivers (temperature-driven development and consumption rates) as well as a tractable epidemiological link to our purely entomological models.Cx. quinquefasciatus larval habitats. Our data and models demonstrate that such density-dependent effects can have dramatic population-level impacts, driving larval productivity and adult Cx. quinquefasciatus population response to rain events. Our study therefore provides a theoretical platform for the study of immature populations and opens the door for more explicit explorations of the impact of larval and adult control in this medically relevant disease vector.Driven by detrital loading through stochastic rain events, density-dependent interactions may be overlooked due to the high larval population levels found within Additional file 1:Annotated code performed in R and used to generate all models presented in this article. (R 30 kb)


Increasing population-based evidence suggests that patients who receive breast conserving surgery (BCS) plus radiotherapy have superior survival than those who receive mastectomy. It is unclear, however, how BCS followed by re-excision is associated with all-cause and breast cancer-specific mortality, and whether the BCS survival advantage is maintained if re-excision is needed. The aim of this study was to investigate the clinical, patient, provider and geographic variation associated with receipt of re-excision surgery, and to examine the relationship between re-excision and all-cause and breast cancer-specific mortality.All women diagnosed with stage I-III breast cancer in Alberta, Canada from 2002 to 2009 were identified from the Alberta Cancer Registry, of which 11,626 were eligible for study inclusion. Type of first breast cancer surgery after diagnosis, subsequent re-excisions within 1 year, surgeon (anonymized), and hospital were obtained from provincial physician claims data. Multilevel logistic regression with surgeons and hospitals as crossed random effects was used to estimate the adjusted odds ratios of re-excision by the factors of interest. Poisson regression models were fitted to compare all-cause and breast cancer-specific mortality by surgical pattern.N = 5659) of patients who initially received BCS. The adjusted odds of re-excision varied significantly by geography of surgery, and by individual surgeon among stage I and II patients beyond the variation explained by the factors investigated (Stage I OR standard deviation (SD) = 0.43; stage II OR SD = 0.39). Patients who were treated with BCS plus re-excision surgery with either mastectomy or further BCS had similar all-cause and breast cancer-specific mortality as those treated with BCS without re-excision.Re-excision surgery was received by 19% (These results suggest that breast cancer patients who are treated with BCS plus re-excision surgery by either mastectomy or further BCS have similar survival as those treated with BCS without re-excision. The significant variation in the likelihood of re-excision by geography and by individual surgeon is concerning, especially given the costs to the patient associated with additional surgery and the financial costs to the health system. Most women diagnosed with early stage breast cancer have the option of receiving either mastectomy or breast conserving surgery (BCS) plus adjuvant radiotherapy, as randomized clinical trials have reported equivalent survival outcomes –3. BCS fIn contrast to the clinical trials reporting equivalent survival outcomes regardless of initial breast cancer surgery choice, population-based studies in the United States and Canada have recently reported poorer survival for patients who receive mastectomy compared to those who receive BCS plus radiotherapy –11. ExisThe purpose of this study was to: 1) investigate the clinical, patient, provider and geographic factors associated with receipt of re-excision surgery and, 2) examine the relationship between re-excision and all-cause and breast cancer-specific mortality.The Alberta Cancer Registry was used to identify women diagnosed with stage I, II or III solid tumor breast cancer  from 20Two sources of data were used, the Alberta Cancer Registry and the Alberta Health Physician claims database. The Alberta Cancer Registry is a population-based cancer surveillance system, that has been awarded the highest level of certification in all years of the study for its high level of completeness and timeliness of data collection and reporting. The Alberta Health Physician claims database collects information about all procedures performed by fee-for-service physicians for billing purposes. All surgeons performing breast cancer resections in Alberta are paid on a fee-for-service basis. All patient information was de-identified, and physical and technological safeguards were put in place to protect the confidentiality of information and the privacy of patients as per the Alberta Health Information Act.N = 194) or nodal status (N = 161) were randomly assigned a value proportionally based on the non-missing information. The following sensitivity analyses were run to test the assumptions: 1) patients with missing tumor size were randomly assigned to be T4 and, 2) patients with missing nodal status were assigned to be N3. Results from the sensitivity analyses did not differ from those of the primary analyses. The North American Association of Central Cancer Registries has awarded the Alberta Cancer Registry the highest level of certification in all years of the study for its high level of completeness and timeliness of data collection and reporting.Demographic, clinical and treatment information were obtained from the Alberta Cancer Registry including: date of and age at diagnosis; geographic region of surgery; cancer stage; tumor size; nodal status; estrogen and progesterone receptor (ER/PR) status; receipt of neo-adjuvant and adjuvant chemotherapy; receipt of hormone therapy; receipt of post-operative radiotherapy; date and cause of death. Geographic region of surgery was categorized into the five administrative health zones of Alberta. Two zones are urban and suburban in population size and density  and three zones are a combination of suburban, rural and remote regions . Cancer stage was determined using the American Joint Committee on Cancer (AJCC) 5th edition staging rules active for years 2002 and 2003, and the 6th edition for years 2004-2009 , 16. PatAlberta Health Physician Claims data were used to identify the first breast cancer surgery after diagnosis and subsequent re-excision procedures up to 1 year after initial surgery; date and type of surgery, surgical hospital; and anonymized physician identifier were obtained. Surgery pattern, i.e. type(s) of breast cancer surgeries received, including re-excision, was classified as BCS without re-excision, BCS plus BCS re-excision, BCS plus mastectomy re-excision and initial mastectomy. Surgeon volume was defined by the number of breast surgeries performed on patients in the study cohort in each surgeon’s highest volume year during the study period, excluding re-excision surgeries. This variable is described in detail elsewhere .Descriptive statistics were calculated for the demographic and clinical characteristics of patients whose initial surgery was BCS by cancer stage. Multi-level mixed effects logistic regression with surgeons and hospitals as crossed random effects  were useTwo additional exclusions were made for the investigation of surgery pattern on overall survival and breast cancer-specific mortality to facilitate the comparison of patients who received standard treatments: 1) patients who did not receive radiotherapy following BCS and 2) patients who received radiotherapy following mastectomy.Descriptive statistics were calculated for the demographic and clinical characteristics of the study patients by surgery pattern. Overall survival and breast cancer-specific mortality of patients by surgical pattern was compared by Kaplan-Meier and cumulative incidence curves, respectively, with deaths from other causes being treated as competing risks in the cumulative incidence analysis. Patients were followed from 1 year post initial surgery until the earliest of date of death or September 30, 2011. Log-rank and Gray’s  test staN = 242), had a second primary breast cancer diagnosis prior to surgery (N = 115), had missing or incomplete billing data (N = 839) or did not have at least 1 year of follow-up following initial surgery (N = 210) were excluded.There were 11,626 eligible patients diagnosed with early stage breast cancer from 2002 to 2009 included in the study; a flow diagram of surgeries received by the eligible cohort is displayed in Fig. BCS without re-excision, BCS plus BCS re-excision, BCS plus mastectomy re-excision and initial mastectomy were received by 39, 4, 5 and 51% of patients, respectively. Older patients were most likely to receive mastectomy, both initially and as a re-excision following initial BCS. BCS plus mastectomy re-excision was more prevalent than BCS plus BCS re-excision in Edmonton and in Central and Southern Alberta, while BCS plus BCS re-excision was more prevalent among patients receiving surgery in Northern Alberta, and performed in approximately equal proportions in Calgary.Patient demographic and clinical characteristics and their associations with re-excision among those who received initial BCS are shown in Table Table Mortality analysis includes 9023 (78%) patients: 517 patients who did not receive radiotherapy after BCS and 2086 patients who received radiotherapy after mastectomy were excluded. Median follow-up time was 4.0 years. A total of 880 (9.8%) patients died during the follow-up period: 406 (4.5%) from breast cancer and 474 (5.3%) from other causes.Figure Adjusted all-cause and breast cancer-specific mortality rate ratios are shown in Table In this study, breast cancer patients who were treated with BCS plus re-excision surgery had similar all-cause and breast cancer-specific mortality as those treated with BCS without re-excision. These results supplement increasing observational evidence that in the population-based context, patients who receive mastectomy have poorer survival than those who receive BCS plus radiotherapy –11, suggThe percentage of patients that received re-excision in this study, 19%, is similar to that found by other population-based studies in England, Ireland and the Netherlands , 25, 26.Re-excision use varied significantly by geography and surgeon among stage I and II patients in the current study. We believe that this is due to the lack of a formal or informal consensus regarding the best definition of negative surgical margins that adequately reduce the risk of recurrence following BCS at the time of this study . A surveSurprisingly, re-excision was not associated with surgeon volume, as other studies have reported this association , howeverRe-excision was also associated with tumor size and, among patients with stage I disease, younger age at diagnosis. BCS performed on a large tumor is a more technically challenging procedure than that performed on a small tumor as it is more difficult to remove a large amount of tissue and simultaneously optimize cosmetic outcome. Additionally, younger women tend to have dense breasts, limiting the ability of the surgeon to accurately assess tumor extent preoperatively . It has The population perspective of the present study is especially valuable as surgical decisions for breast cancer are based on various factors including patient preference and contraindications to radiotherapy. It is therefore of clinical interest to investigate surgical patterns in a population-based and unselected series of patients with minimal bias caused by variation in access to care often present in alternatively-funded health care systems. Multi-level modeling was used to account for the hierarchical data structure and provide estimates of the level of variation within surgeons and hospitals. Limitations of the current study are largely due to the nature of the administrative datasets; information about margin status, breast density, recurrence and prognostic factors such as comorbidities, lifestyle factors or socioeconomic status were not available. Lack of these variables may introduce confounding by indication if higher risk patients are more likely to receive mastectomy. Additionally, longer follow-up may be necessary to detect survival differences between the surgery pattern groups.The current results suggest that patients who receive BCS plus re-excision with either BCS or mastectomy have similar survival outcomes as patients who receive BCS without re-excision. Although not possible in the current study, investigation of recurrence rates and surgical patterns, would be of interest. Given the increasing population-based results favoring initial treatment with BCS plus radiotherapy over mastectomy, and considering that mastectomy is a more invasive procedure, fear of additional surgery should not be a reason to receive initial mastectomy. We suggest greater efforts towards educating and encouraging women to receive initial BCS rather than mastectomy when appropriate. This may be particularly important in the face of increasing mastectomy rates , 33, incAdditionally, although the survival of patients who received re-excision was not significantly different from those who received a single BCS procedure, the significant variation in the likelihood of re-excision by geography and by individual surgeon is concerning, especially given the costs to the patient associated with additional surgery and the financial costs to the health system. We suggest targeted education efforts for surgeons on the recent consensus guidelines to facilitate increasing surgeon uptake, which may help to reduce variation and prevent unnecessary re-excision. Re-evaluation of re-excision rates in Alberta in the coming years will be of interest.


Previous studies have demonstrated that altered states of consciousness are related to changes in resting state activity in the default-mode network (DMN). Anatomically, the DMN can be divided into anterior and posterior regions. The anterior DMN includes the perigenual anterior cingulate cortex and other medial prefrontal cortical regions, whereas the posterior DMN includes regions such as the posterior cingulate cortex (PCC) and the temporal parietal junction (TPJ). Although differential roles have been attributed to the anterior and posterior DMN regions, their exact contributions to consciousness levels remain unclear. To investigate the specific role of the posterior DMN in consciousness levels, we investigated 20 healthy controls  and 20 traumatic brain injury (TBI) patients  whose brain lesions were mainly restricted to the bilateral frontal cortex but retained a well-preserved posterior DMN  and who exhibited varying levels of consciousness. We investigated the intra- and cross-functional connectivity strengths (FCSs) between the right/left PCC and the right/left TPJ and their correlation with consciousness levels. Significant reductions in both the intra- and cross-hemispheric FCSs were observed in patients compared with controls. A significant correlation with consciousness levels was observed only for the cross-hemispheric PCC-TPJ FCS but not for the intra-hemispheric PCC-TPJ FCS. Taken together, our results show that the cross-hemispheric posterior DMN is related to consciousness levels in a specific group of patients without posterior structural lesions. We therefore propose that the PCC may be central in maintaining consciousness through its cross-hemispheric FC with the TPJ. In addition, recent studies of individuals in altered states of consciousness, such as under anesthesia or in an unresponsive wakefulness/vegetative state (UWS/VS), have reported alterations in the resting state functional connectivity in the DMN10. Despite several studies, the exact functional role of the DMN specifically regarding consciousness remains unclear.The default-mode network (DMN) is one of the central resting state networks in the brain12. Although differential roles have been attributed to the anterior and posterior DMN regions13, their exact functions remain unclear. Studies of altered consciousness have observed changes in both the anterior and posterior DMN14 (see above). This finding raises questions regarding the exact and possibly distinct roles of the anterior and posterior DMN regions and their specific contributions to consciousness.Anatomically, the DMN can be divided into anterior and posterior regions. The anterior DMN includes the perigenual anterior cingulate cortex and other medial prefrontal cortical regions, whereas the posterior DMN includes regions such as the posterior cingulate cortex (PCC) and the temporal parietal junction (TPJ)15. Moreover, recent studies have suggested that the PCC may be central for consciousness17. We therefore hypothesized that the levels of hemispherical functional connectivity in the PCC may be related to consciousness levels.The posterior midline regions, such as the PCC, exhibit high degrees of resting metabolism19, but its relation to consciousness levels remains unknown. In addition, the specific effects posterior DMN, including the PCC, and their relation to the levels of consciousness are not well defined. Therefore, the current study sought to analyze both the intra- and cross-hemispheric functional connectivity between the PCC and the TPJ in a unique sample of patients with selective frontal lesions but no anatomical structural lesions in the posterior DMN and who exhibited varying levels of consciousness. Based on our own20 and previous23 results, we hypothesized that despite the absence of structural lesions the PCC-TPJ FCS may be reduced and related to the level of consciousness.The difference in the function between the left and right hemispheres has been previously discussedThe aim of our study was to identify the impact of resting state functional connectivity in the posterior DMN  on the level of consciousness in a sample of subjects without lesions in the posterior DMN. We investigated a sample of subjects with brain lesions primarily restricted to the bilateral prefrontal cortex but with well-preserved posterior DMN regions, e.g., the PCC and the TPJ, and varying levels of consciousness.The experiment included 20 healthy control subjects  and 23 bilateral frontal injury cases . All human studies were approved by the Ethics Committee of Huashan Hospital and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All patients provided their informed consent prior to inclusion in this study. These patients, who were enrolled when they were physically stable and did not received any consciousness-related drugs within one week, mainly suffered from bilateral frontal contusions with an intact posterior DMN . Table All subjects had no history of drug or alcoholic addiction, cerebrovascular or mental diseases, nor any congenital brain diseases .A Siemens Magnetom Verio 3.0 T MRI scanner  with a standard head coil was used to scan the patients in the Department of Radiology of Huashan Hospital, Shanghai, China. The gradient-echo EPI images of the whole brain parameters were set as follows: 33 slices, repetition time/echo time [TR/TE] = 2,000/35 ms, slice thickness = 4 mm, field of view = 256 mm, flip angle = 90°, image matrix: 128 × 128). In total, 200 scans (406 seconds) were acquired in the resting run. In addition, high-resolution anatomical images were acquired for all subjects. T1-weighted magnetization-prepared rapid-gradient echo (MPRAGE) sequences were acquired through the sagittal plane with the following parameters: TR = 2300 ms; TE = 2.98 ms; TI = 900 ms; flip angle = 9°; matrix size = 256 × 240; slice number = 176; field-of-view (FOV) = 256 × 240 mm; acquisition averages = 1; scanning time = 301s. Patients’ CT scans were collected for detecting and identifying cerebral lesions. If the quality of the fMRI data were not satisfactory, such as being confounded by excessive head movements, a scan was performed again on a subsequent day.24.All subjects’ MRI scans were acquired under resting states. For each patient, consciousness assessment were performed according to the standardized Glasgow Coma Scale (GCS) and CRS-Rhttp://afni.nimh.nih.gov/afni). Due to the severe injury/lesions to the frontal lobes, spatial normalization was not performed, and the individual analysis was conducted. The preprocessing steps included head motion correction25, slice timing correction, spatially smoothing with a 6 mm full-width at half-minimum (FWHM) isotropic Gaussian kernel and temporal normalization. Scans with head motion greater than 0.5 mm of displacement9 in the x, y or z direction or 1.5 degrees of any angular motion throughout the course of the scan were excluded27. Any subjects with less than 100 volumes were completely excluded. According to those standards, 3 patients  were excluded from the experiment who corresponded to less than 100 volumes. The resting functional data were then filtered with a band-pass filter reserving signals between 0.01 and 0.1 Hz, which is regarded as the main reflection of neural fluctuations28.All MR imaging data were analyzed using Analysis of Functional Neuroimage (AFNI) software .After the head motion correction procedure, the magnitude of the head motion at each time point for 6 parameters (3 for shift and 3 for rotation) was obtained for each subject. The averaged head motion parameters for shift and rotation were then calculated via the method reported in the studies by Zang31. After the PCC seed was chosen, the PCC seed-based functional connectivity map was acquired. Then, the correlation coefficients R of the PCC-TPJ (see the definition of TPJ below) for individual subjects were extracted and transformed to Fisher’s Z. The hemispheric Z scores, which represented the seed-based hemispheric FC strength indicating the relationships of lPCC-lTPJ and lPCC-rTPJ (from the left PCC seed-based map) and rPCC-lTPJ and rPCC-rTPJ (from the right PCC seed-based map)  was used, as calculated by AFNI. The intra- and cross-hemispheric differences of the right/left PCC seed-based FCSs have not been previously investigated in frontal TBI patients.14; (2) posterior to corpus callosum splenium at 0.5 cm; (3) perpendicular to the parietooccipital sulcus at 0.5 cm; (4) near the midline of the brain; (5) spheres with radii of 0.5 cm are drawn for the left PCC (lPCC) and right PCC (rPCC) seed regions.As the incomplete brain structure, the left/right hemispheric PCC seeds were set individually for both the control and patient groups according to the following criteria: (1) the posterior cingulate cortex is the backmost part of the cingulate cortex, lying behind the anterior cingulate cortex. The PCC forms part of the posteromedial cortex with the retrosplenial cortex (Brodmann areas 29 and 30) and the precuneus (located posterior and superior to the PCC)After the PCC seed-based FC maps were calculated, we defined the TPJ ROI (Region of Interest) according to the FC map with the following criteria: (1) the anatomical temporoparietal junction lies in the region between the temporal and parietal lobes near the Sylvian fissure. Specifically, it is composed of the inferior parietal lobule and the caudal parts of the superior temporal sulcus; (2) Also, according to the hemispherical PCC based functional connectivity map, the highest correlation cluster  was included in TPJ ROI; (3) spheres with radii of 0.5 cm are drawn in hemispherical TPJ regions as the left TPJ (lTPJ) and right TPJ (rTPJ) ROIs, respectively. All values shown for the functional connectivity in all the posterior regions are ROI-based rather than voxel-based. All of the determined ROIs were double checked by the second (R.D.) and second to last author (X.W.) of the paper.Between- and intra-group multiple comparisons of the intra- and cross-hemispheric PCC-TPJ FCSs from patients and healthy subjects were performed. Then, an intra- and between-group repeated-measures ANOVA was conducted to detect differences among the PCC-TPJ FCS patterns of the healthy and patient groups. To investigate the relationship between the PCC-TPJ FCS and the level of consciousness, we conducted two types of analyses. First, the patient group was divided into three subgroups for the levels of consciousness according to the high (GCS = 12–15), medium (GCS = 9–11), and low (GCS = 3–8) GCS scores. Due to the insufficient number of patients in the low group, the low and medium consciousness groups were combined (n = 9) for comparison with the high consciousness group (n = 11) using an independent sample t-test. Second, we conducted Spearman’s correlation analyses between the PCC-TPJ FCSs and the GCS scores and between the PCC-TPJ FCSs and the CRS-R scores. A partial correlation was conducted with covariates, including head motion (Mt) and grey and white matter volumes. All the aforementioned results were thresholded at P < 0.05.33; therefore, it is difficult to determine whether the origins of their signals are from conserved neurons. In patients with frontal lobe injuries, the reserved posterior regions may more accurately reflect changes in the DMN within DOC (Disorder of Consciousness) states.A common way to detect the DMN and consciousness alterations of TBI patients is using the whole brain functional map, which may include the trauma regions and their peripheral areas. Previous studies have shown that these regions are located within anatomically damaged areas that contain astrocytes undergoing proliferation and repairAn analysis of the FCSs between ROIs was performed . A 2-way repeated-measures mixed ANOVA with pairs of regions as within-group factor and a between-group factor was conducted and revealed differences between the controls and patients , significant differences among 4 types of PCC-TPJ patterns , and no significant interaction effect of the PCC-TPJ patterns and the subject groups . Then, the T-test with Bonferroni multiple-comparison correction was used to detect differences among all of the PCC-TPJ FCS patterns between controls and patients. This test revealed significant decreases in both the intra- and cross-hemispheric resting state FCSs of the patient group  patients (n = 11) were compared with a subgroup of medium- (GCS = 9–11) and low- GCS = 3–8) scoring patients (n = 9). This result revealed significantly lower PCC-TPJ FCSs in the medium/low group than in the high group. A highly significant difference was found for the cross-hemispheric PCC-TPJ FCS between the two subgroups, whereas a significant difference was not detected in the intra-hemispheric PCC-TPJ FCS , grey and white matter volumes) were conducted between the PCC-TPJ resting state FCS and the GCS scores Table . As befoIn the current study, we investigated the intra- and cross-hemispheric resting state functional connectivities in the posterior DMN in a unique sample of bilateral frontal-injured patients with varying levels of consciousness and with no underlying anatomo-structural lesions in the posterior DMN. The main findings are as follows: (i) significantly decreased resting state PCC-TPJ FCS was observed in the frontal lesion patients compared with the healthy subjects; (ii) there was a difference between the left intra- and cross-hemispheric FCSs in the patient group; and (iii) a significant correlation between the cross-hemispheric PCC-TPJ FCSs and the level of consciousness was observed, whereas such a correlation was not observed for the intra-hemispheric PCC-TPJ FCSs. By enrolling a unique group of TBI patients with no posterior DMN lesion, our study showed for the first time the functional impact of the posterior DMN on the level of consciousness with a special role in the cross-hemispheric PCC-TPJ resting state functional connectivity.We therefore suggest that cross-hemispheric FCS (as distinguished from intra-hemispheric FCS) in the posterior DMN regions between the PCC and the TPJ may play a particularly central role in contributing to the level of consciousness.35. Our study extends upon these previous results by showing cross-hemispheric PCC-TPJ changes in a special group of patients with selective frontal lesions but without structural posterior DMN lesions. While controlling for various covariates , significant correlations were detected between the cross-hemispheric resting state PCC-TPJ functional connectivity and the level of consciousness including a higher cross-hemispherical FCS (lPCC-rTPJ and rPCC-lTPJ) and a higher level of consciousness. By contrast, we did not find any such correlation between the intra-hemispherical FCS and the level of consciousness.In our study, the results demonstrated that the posterior DMN could be affected by frontal lesions and reflect the levels of consciousness, which is in line with previous studiesThe relation of the PCC-TPJ FCS with the MPFC and the relation between the anterior and posterior regions of the DMN remain unclear. All our patients showed bilateral MPFC lesions; therefore, we were unable to subgroup our patient sample according to different degrees of MPFC lesions. Future studies should therefore test the impact of the MPFC on PCC-TPJ, such as during anesthesia, when results would not be confounded by lesions to either the MPFC or the PCC.36. Our results extend these observations by showing a specific role of cross-hemispheric PCC-TPJ FCS in the level of consciousness: the PCC’s communication with the TPJ in the contralateral hemisphere seems to be central for maintaining the level of consciousness. This finding is even more remarkable given that both the intra- and cross-hemispheric resting state PCC-TPJ FCSs were reduced in the TBI group, in accordance with the whole-brain FCS decrease observed in DOC patients in previous studies38. This result suggests a specific role for cross-hemispheric PCC-TPJ resting state functional connectivity in maintaining (or deteriorating) and reflecting the level of consciousness.The right and left TPJ are related to different functions, such as self-produced actions and attention in the right TPJ and language cognition, processing, and comprehension of both written and spoken language in the left TPJ39. These studies showed PCC signal changes in DOC patients conducting auditory tasks9, a link between temporal variability and signal synchronizations that include PCC-region signals during wakefulness that is disrupted during anesthesia40, and decreases in the PCC signals in subjects undergoing hypnosis41. Our results are consistent with these results and extend them with regard to a specific role of cross-hemispheric FC of the PCC to the TPJ. Moreover, because our subjects exhibited exclusive frontal lesions without lesions to posterior DMN regions, the cross-hemispheric PCC-TPJ effects must be considered functional rather than based on structural lesions.The PCC is a core region of the posterior DMN located in both hemispheres, which has been shown to be related to consciousness in previous studies42, which have been shown to be associated with consciousness44. Due to their anterior lesions, the cingulate bundle, which passes from anterior to posterior DMN regions, may be affected. This notion requires future investigations via methods such as diffusion tensor imaging (DTI). However, our results clearly demonstrate the importance of cross-hemispheric interactions and cross-hemispheric functional connectivity38 in TBI patients and in subjects with altered states of consciousness in general.However, the exact origin of the disrupted PCC-TPF FCS remains unclear. First, it may be due to micro-lesions in the PCC hemispheric connecting fibers. Second, the origin may also be related to disturbed functional connectivity of the PCC and the TPJ to the anterior DMN regions, such as the MPFC regionsIn conclusion, we investigated a unique TBI patient group with varying levels of consciousness and absent anatomo-structural lesions in the posterior DMN regions. This made our group of subjects particularly suitable for investigating the role of the posterior DMN in consciousness. We observed significant decreases in both intra- and cross-hemispheric PCC-TPJ resting state functional connectivity in the TBI patient group compared with the healthy subjects. However, only cross-hemispheric (rPCC-lTPJ and lPCC-rTPJ) resting state FCSs (but not intra-hemispheric resting state FCSs) predicted the level of consciousness in the TBI patient group. This result suggests a special, although yet unclear, role of cross-hemispheric posterior DMN resting state FCS in maintaining consciousness.Author contribution statement


SWC format, thus ensuring full back-compatibility with popular visualization and modeling tools. We then deploy the combined multichannel/time-varying reconstruction system on developing neurons in live Drosophila larvae by digitally tracing fluorescently labeled cytoskeletal components along with overall dendritic morphology as they changed over time. This same design is also suitable for quantifying dendritic calcium dynamics and tracking arbor-wide movement of any subcellular substrate of interest.Several efficient procedures exist to digitally trace neuronal structure from light microscopy, and mature community resources have emerged to store, share, and analyze these datasets. In contrast, the quantification of intracellular distributions and morphological dynamics is not yet standardized. Current widespread descriptions of neuron morphology are static and inadequate for subcellular characterizations. We introduce a new file format to represent multichannel information as well as an open-source Vaa3D plugin to acquire this type of data. Next we define a novel data structure to capture morphological dynamics, and demonstrate its application to different time-lapse experiments. Importantly, we designed both innovations as judicious extensions of the classic  In parallel to continuous improvements in labeling and imaging techniques, methods to trace axonal and dendritic branching evolved from pencil-on-paper to digital encoding of tree origins, bifurcations, and terminations3. Even though two-dimensional (2D) analyses of neuron morphology have remained in practice4, the more complete and realistic three-dimensional (3D) reconstructions became the standard in the field5. As the number of scientific publications describing 3D digital tracings continued to increase, NeuroMorpho.Org emerged as a popular electronic repository to store, annotate, publicly share, and freely reuse these labor-intensive datasets6. Although individual neuroscience labs upload 3D reconstructions to NeuroMorpho.Org in more than 20 different formats depending on the specific reconstruction system they use, all data are converted to, and can be downloaded in, a common lingua franca, the SWC file descriptor7. The open availability of neuronal morphology digitally reconstructed in this form from a vast array of model systems, experimental preparations, anatomical regions, and cell types enabled a diverse array of secondary studies. Among the most flourishing applications are comparative morphometric analyses8, electrophysiological simulations9, large-scale biophysically-detailed modeling10, and algorithmic generation of virtual neurons12.Neuroscientists have long recognized the importance of neuronal structure in defining circuit function. Ramón y Cajal began by drawing the complex shape of Golgi-stained neural arbors13, the existing representation of neural structure has two main limitations. First, 3D reconstructions describe the overall morphology of neurons, but no information on intracellular substrates. Rapid technological progress in serial immuno-staining14, genetic fluorophore engineering15, and spectral unmixing16 now allow simultaneous labeling of multiple subcellular components, requiring a co-evolution of the digital representation system. Second, current reconstructions are static, and do not incorporate temporal dynamics of neural structure. In the past, neuroinformatic tools have been developed to deal with subcellular17–19 and dynamic21 neural data. Nevertheless, major advancements in live imaging techniques22–24 necessitate an extension of the neuroanatomical file descriptors to annotate morphological changes over time. In both cases, the ideal data structure should capture these additional dimensions flexibly and precisely while preserving the intuitive simplicity of the original SWC format.While the advent of high-throughput automated tracing has dramatically expanded the sheer volume of experimental dataESWC) and the corresponding Vaa3D25 plugin to acquire multi-signal reconstructions that incorporate subcellular information simultaneously with the overall morphology of the neuron. This application repurposes the ESWC extension previously introduced in Vaa3D to explicitly incorporate specific morphological features for faster computation26. We then introduce a novel data structure (SWCX) to represent temporal branching dynamics. These next-generation neural tracing systems are well suited for studying the cytoskeletal effectors of neural growth and the genetic programs that govern cytoskeletal dynamics27. Specifically, fluorescently labeled multi-signal live images of developing neurons from Drosophila larvae can aid in elucidating the biochemical mechanisms underlying the known morphological diversity of neuron types28. We thus demonstrate how multi-signal and time-lapse reconstructions may be combined to describe subcellular structural dynamics in this genetically tractable experimental system.Here we present an expanded multichannel file format  neuronal trees  the volumetric fraction in which the signal is present, measured as the proportion of voxels that are above threshold; (ii) the mean signal intensity of those voxels; and (iii) their standard deviation (s.d.). While the mean signal intensity represents the overall local concentration of the labeled substrate, the ratio above threshold and s.d. help distinguish between diffused and punctate signals independently for each channel. A compartment with ratio near 1 and low s.d. signifies homogenous substrate distribution, whereas a ratio close to 0 and high s.d. indicates strong punctate signal expression. Thus, if two subcellular components are simultaneously imaged, the ESWC description will consist of 13 values for each tracing point: 7 to reconstruct the arbor morphology and 3+3 to quantify each of the two parallel channels.Since the morphology of neuronal trees corresponds to the continuous bounds of their cytosolic membrane, the 3D location and thickness of each branch are typically traced from the image stack of the membrane label. The spatial distribution of intracellular constituents, in contrast, is often non-uniform and discontinuous. Thus, when an additional imaging channel reports a distinct subcellular element, its staining intensity can vary within a given branch based on the local concentration. To capture such multi-signal information in digital reconstructions, we designed an extension of the multichannel_compute plugin for the Vaa3D software suite (Vaa3D.org). Specifically, this plugin automatically generates the ESWC file from the multichannel image stacks and the corresponding standard SWC file (see Methods). In addition to outputting the ESWC file, the Vaa3D multichannel_compute plugin also saves a backward-compatible version of the SWC file that can be opened using any existing SWC viewer and other legacy tools. The beginning of this file is identical to the original SWC input, but the signal information from all additional channels is appended as a pseudo-comment at the end. In principle, this system can quantify any number of signals by adding three values for each imaged channel to every node. As an illustrative demonstration, we apply this multi-channel reconstruction to simultaneously quantify the distributions of microtubule (MT) and F-actin in a Class I da neuron from the fruit fly larva . Specifically, the subcellular components are displayed as frustums internal to the overall external structure, representing the volumetric fraction occupied, while signal intensity is coded by color  also constitutes a (different) extension of the SWC format. An SWCX file  dynamics within a growing dendritic branch from a mature Class IV da neuron from the fruit fly larva sensory system  or the number of time points captured (SWCX). Such information can be provided in the header of the files along with other useful metadata.The y system . Here, g30. Alternatively, the volume occupied by a neuron structure can be defined by a collection of vertices connected to polygons. Such mesh-like description is often employed in electron microscopy as well as in detailed numerical simulations of molecular diffusion31. This representation, however, is overly data-intensive for the majority of morphological studies and computational models. A more efficient method of capturing overall neural morphology represents branching arbors as sequences of interconnected frustums. The resulting digital reconstructions are especially suitable to describe relatively lower resolution image stacks from light microscopy. This basic data structure has remained almost unaltered in the last forty years30 as further efforts towards technical improvements have primarily focused on automating the tracing process3. Based on estimations from NeuroMorpho.Org’s literature collection, more than 180,000 neurons have been digitally reconstructed to date from at least forty species and over two hundred anatomical regions.The two-dimensional neural drawings by Cajal and his pupils not only demonstrated the remarkable diversity in neuronal structures across and within brain regions and animal species, but also revealed fundamental functional principles such as directional information flow and specificity of neural connectivity. Several types of mathematical descriptors have since been developed to represent axonal and dendritic morphology. Early neuron tracing systems first listed the tracing points in ASCII files as separate text lines, recording local branch position and thickness as well as the topological type SWC format, introduced almost two decades ago7, remains highly popular due to its unsurpassed simplicity. Over 70,000 neuronal reconstructions are freely available in this format from the centralized repository NeuroMorpho.Org33, and dedicated open-source analysis tools have been also developed for the SWC system34. The main rationale for expanding the SWC format instead of any other file system to annotate temporal and subcellular information is precisely this existing (and growing) wealth of available resources3. Instead of coming up with a completely new design, extending the SWC system enables the research community to continue leveraging a wide variety of data acquisition, analysis, and modelling tools. The ESWC and SWCX file structures also largely preserve the simplicity and readability of SWC. Although here for the sake of clarity we have used separate file formats (ESWC and SWCX) to describe these two conceptually distinct extensions of the existing neural description system, in the future it may become convenient to merge the two formats into a single extension along with additional expansions.The 35. Combining paired recording with array tomography enabled researchers to study the interrelation between functional plasticity and molecular composition of synapses36. The positions of synapses have been detected with high spatial accuracy using mGRASP37. Integrated systems for two-photon imaging and photo-stimulation are well suited for systematic interrogations of structural plasticity38. Low-intensity live-imaging of fluorophore-tagged subcellular protein complexes allow long-term tracking of mitochondrial trafficking in neurites39. Intensity of light emitted from calcium sensitive dyes can be measured from multiple distinct locations of the neural arbor across several time points40. Calcium imaging combined with whole-cell patch clamp recording has revealed that spike back-propagation triggers a path distance-dependent calcium rise in dendritic trees41. Advanced genetic toolkits also allow for optogenetic activation of single neurons followed by measurement of activity in functionally connected circuits42. Combination of live-imaging and electron microscopy has revealed the arbor-wide locations of synapses and their ultrastructural architecture43. Bimodal dendritic plasticity dependent on visual stimulation has also been observed through live-imaging44. All the above examples and many more are in principle suitable for digital reconstruction leveraging the novel data structures introduced here.The combination of multi-signal and time-lapse digital reconstructions in principle allows the subcellular quantification of any neuronal property that can be captured over time through light-microscopic imaging, including biochemical concentrations, ion channel locations, and organelle movement, among others. Progress in multi-signal/time-lapse imaging techniques have already started to yield new findings on the subcellular and molecular organization of neurons. For instance, super resolution imaging has revealed the arbor-wide distribution of proteins associated with post-synaptic receptors17–19 and time-lapse21 neural images. However, the community needed a standard descriptor of neuro-structural dynamics45 capable of adding multiple dimensions of information. We demonstrated the newly introduced multichannel (ESWC) and time-lapse (SWCX) data structures in da neurons from the Drosophila larva. Measuring arbor-wide quantities of subcellular substrates in this model system may reveal the influence of individual molecules on mature arbor morphology. Time-varying reconstructions enable the identification and temporal linking of dynamic changes. Combining the multi-signal and time-varying systems allow one to measure the exact changes in cytoskeletal quantity within branches as they elongate or retract across the whole arbor, at the limit of light-microscopic resolution. Subcellular concentrations of growth mediating cytoskeletal proteins can be used as fundamental determinants of dendritic growth in computational simulations, and time-varying reconstructions can be used to improve and validate data-driven models. This type of dynamic data can then be analyzed, visualized and reproduced via simulation46. Notably, time-lapse imaging can capture information across a broad range of temporal scales depending on the dynamics of the biological phenomena under consideration. Dendritic spines and axonal varicosities can turn over in minutes while arbor structures change over weeks. The SWCX system is suitable to represents any and all time scales as illustrated in this report.Significant progress has been made in the annotation systems for multi-signal47. Connectivity columns can be added by annotating pre-synaptic and post-synaptic neuron pairs. Useful anatomical information also includes the location and orientation of reconstructed neurons relative to each other or to tissue layer boundaries. This additional knowledge, if available, can also be encoded in the header of the new augmented files. While the data trends in NeuroMorpho.Org suggests that most of the reconstructions in the near future will still be in the basic 3D static format, we predict that the number of new time-lapse and multichannel datasets will soon start to increase. Availability of simple file formats for these data may also facilitate the development of new tools and resources for the analysis of live and multi-signal neural images48.A complementary augmentation in neuron description not addressed in the present work is the annotation of circuit connectivityESWC reconstructions were generated starting from the multi-channel image data and the standard SWC reconstructions. First the basic SWC file was created by tracing the overall neuronal structure in neuTube49. This file was then used as input in the multichannel_compute plugin of Vaa3D25 along with the image stacks for each channel. The plugin interface asks for a primary channel, a secondary channel, and their intensity thresholds. Within each compartment (frustums defined by the basic SWC file), the plugin then identifies the voxels with intensities above the input threshold for the primary channel  and then checks the intensity of the same voxels for the secondary signal. We ran the plugin twice, once with microtubule and then with F-actin as the secondary signal, using 15  as the voxel intensity threshold (any voxel below 15 is not considered). The plugin outputs two multichannel files. The first file details the multichannel data in the ESWC format with three additional columns for each channel, annotating (1) fraction of voxels above threshold, (2) mean intensity, and (3) standard deviation of intensity. The second file, in a back-compatible SWC format with the regular 7 columns, appends the fraction and mean of subcellular channels at the end of the basic neuron tree after a #CHANNELSWC tag. The Vaa3D multichannel_render plugin uses the simple multichannel file as input and generates a multichannel render file as output, representing the secondary signal distribution as a collection of internal frustums, and the overall morphology as the connected external frustums. The intensity values for each frustum can be color-coded using the Color_render_eswc_feature plugin, where the input parameters are the lower and upper limits of the intensity values.Multi-signal SWCX files for both examples of time-varying reconstructions illustrated in SWC file using neuTube. We then opened the image stack from the 2nd time point along with the 1st time point SWC in neuTube and started annotating the structural changes. Since neuTube only handles the standard SWC format, we temporarily repurposed the neurite type column (2nd field) of the SWC file to tag elongation, local scaling, retraction, re-emergence, and movement events in a ‘time-coded’ manner. The header of the SWC file is used to mark global changes across time points such as overall skeleton scaling. Next we compared the original SWC file (representing the first time point) and the edited SWC file . The ordering of nodes remains unchanged across all time points, and new branches are simply inserted after their corresponding parent nodes in case of terminal extension, and after the entire subtree in case of interstitial extension. This allows for a straightforward assignment of temporal correspondence for all nodes present in both time points. After this step we repeated the process by mapping the (edited) SWC file from the 2nd time point onto the image stack from the 3rd time point and continue until the last time point. At the end, we consolidated the series of annotated SWC files (one for each consecutive pair of time points) into a final SWCX file with a time column for each time point and with numeric codes for each kind of structural change. Additional columns for each time point are allocated to explicitly update the 3D coordinates, radius, and structural connectivity of each node . The intermediate dynamic SWCX files from each time point can be used as input for the multichannel_compute_plugin along with the multi-signal image stacks to combine temporal and sub-structural information, as shown in We created the Drosophila stocks were reared at 25 °C on standard cornmeal-molasses-agar media. The following fly strains were used in the study GAL4221,UAS-mCD8::GFP (Class I); GAL419-12,UAS-mCD8::GFP (Class III); GAL4477, UAS-mCD8::GFP/CyO,tubP-GAL80; GAL4ppk1.9, UAS-mCD8::GFP (Class IV); UAS-GMA::GFP; GAL4221,UAS-mCherry::Jupiter; UAS-LifeAct-Ruby. Fluorescently labeled da neurons from age-matched third instar larvae were imaged via in vivo confocal imaging using previously established protocols21. Briefly, larvae were placed on a microscope slide, immersed in 1:5 (v/v) diethyl ether:halocarbon oil 700 and covered with a 22×50 mm coverslip. Neurons were visualized on a Zeiss LSM 780 confocal microscope. Three-dimensional z-stacks were collected using a 20X/0.8 N.A. air objective at step-size of 1.0–2.0 μm and 1024×1024 resolution. For time-lapse imaging, images were acquired in time series mode with a time interval of 2 min between frames at a step-size of 2 μm and 1024×1024 resolution.How to cite this article: Nanda, S. et al. Design and implementation of multi-signal and time-varying neural reconstructions. Sci. Data 5:170207 doi:10.1038/sdata.2017.207 (2018).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In the past decade we have been proposing a temporal sequence of events in the HCV-infected cell whereby the primary alteration is localized at the mitochondria-associated ER membranes and causes release of Ca2+ from the ER, followed by uptake into mitochondria. This ensues successive mitochondrial dysfunction leading to the generation of reactive oxygen and nitrogen species and a progressive metabolic adaptive response consisting in decreased oxidative phosphorylation and enhanced aerobic glycolysis and lipogenesis. Here we resume the major results provided by our group in the context of HCV-mediated alterations of the cellular inter-compartmental calcium flux homeostasis and present new evidence suggesting targeting of ER and/or mitochondrial calcium transporters as a novel therapeutic strategy.Chronic hepatitis C is characterized by metabolic disorders and by a microenvironment in the liver dominated by oxidative stress, inflammation and regeneration processes that can in the long term lead to liver cirrhosis and hepatocellular carcinoma. Several lines of evidence suggest that mitochondrial dysfunctions play a central role in these processes. However, how these dysfunctions are induced by the virus and whether they play a role in disease progression and neoplastic transformation remains to be determined. Most  Approximately 80% of acutely infected individuals develop chronic infection which may progress to cirrhosis in 2–20% after 20 years and in 15–30% after 30 years. Once cirrhosis is established, the risk of hepatocellular carcinoma (HCC) development is 1–5% per year  contribute to the virus particle, the others  are functional proteins necessary for replication and assembly of the virion  which is anchored on the mitochondrial outer membrane and that after binding of RIG-1 provides a recruiting platform for a number of factors whose activation leads to expression of interferon-β  overload; (ii) dissipation of the mitochondrial membrane potential (ΔΨm), which correlates with inhibition of cell respiration and complex I (NADH dehydrogenase) activity; (iii) overproduction of reactive oxygen and nitrogen species (RO/NS). Time-resolved analysis demonstrated that mtCa2+ overload was the earliest mitochondria-related alteration following induction of HCV protein expression   or Ru360, both inhibitors of the MCU   since inhibition of either of them prevents and reverses the HCV protein-mediated mitochondrial alterations.Combination of all the above reported observations supports a pathogenetic model for HCV infection whereby a self-nourishing mechanism is activated, consisting in positive feed-back loops initiated by the entry of Can Figure . In such2+ flux homeostasis in the interplay between HCV and the host cell is also underlined by the presence in the HCV proteins of the viroporin p7  inhibition of respiratory chain activity and oxidative phosphorylation; (iii) reduction of membrane potential; (iv) overproduction of reactive oxygen species. The effects of amantadine were observed within 15 min following drug administration and confirmed in Huh-7.5 cells transfected with the infectious full-length HCV genome. However, these effects were also observed in cells expressing subgenomic HCV constructs, indicating that they are not mediated or only in part mediated by p7. Single organelle analyses carried out on isolated mouse liver mitochondria demonstrated that amantadine induces hyperpolarization of the membrane potential , HCV-infected cells did not show evident signs of sufferance or bioenergetic failure. Indeed, the difference in growth rate and viability of both HCV-induced U2-OS cells and HCV-infected Huh-7 cells was negligible as compared with control cells. Accordingly, the cellular ATP level was unaffected, if not increased, in HCV-induced U2-OS cells grown in glucose-containing media    at the same time tunes the consequent effects evading premature cell-death signaling of the host cell.A recently emerged property of HCV is the enhanced mitophagy in the host cell   and a PDH phosphatase (PDP)  biosynthesis is likely to occur concurrently with FAO dampening. Consistently, we found up-regulation of the FAS rate-limiting enzyme acetyl CoA carboxylase  in such a multistep process, HCV infection can progress to different clinical outcomes including steatosis, fibrosis, HCC (Figure In conclusion, the emerging strategy put in action by HCV is consistent with a fine rewiring of host cell metabolism. This is achieved by depressing mitochondrial OxPhos while fostering glycolysis which provides, in addition to energy, precursors for biosynthetic processes. A major and perhaps germinal event appears to be a deregulation of CaC Figure .Importantly, the development of drugs selectively targeting ER and/or mitochondrial calcium channels might represent a potential strategy in support of standard HCV therapies.RS: Carried out the experiments, analyzed the results; CP: Designed and supervised the study, carried out the experiments; DM: Provided expertize, samples and critical feedback, assisted in writing the paper; NC: Designed the study, supervised the project, wrote the paper.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.