The effects of intravenous fluid therapy on fluid compartments and hemodynamics of the human body remain enigmatic. We therefore tested the efficacy of bioimpedance spectroscopy in a crossover study, where 15 males received 0.5 ml/kg/min ELO-MEL-isoton (osmolarity = 302 mosmol/l) during 60 minutes, or nothing at all. In group “Fluid”, fluid load increased from −0.2 ± 1.0 l extracellular volume at baseline to its maximum of 1.0 ± 0.9 l in minute 70, and remained continuously elevated throughout minute 300. In group “Zero”, fluid load decreased from 0.5 ± 1.1 l at baseline to its minimum of −1.1 ± 1.1 l in minute 300. In group “Fluid”, intracellular volume decreased from 26.8 ± 3.9 l at baseline to its minimum of 26.0 ± 3.9 l in minute 70, and remained continuously decreased throughout minute 300. In group “Zero”, intracellular volume increased from 26.5 ± 3.8 l at baseline to its maximum of 27.1 ± 3.9 l in minute 120, and decreased thereafter. In group “Fluid” compared to “Zero”, systolic blood pressure was significantly higher, from minute 50–90. In conclusion, intravenous fluid therapy caused a clinically meaningful, sustained increase in fluid load, and a decrease in intracellular volume. These data raise interest in studying fluid administration by the gastrointestinal route, perhaps even when managing critical illness. Essential decisions, such as amount of infusion and fluid type to be administered6 are frequently made in absence of clinical knowledge on the desired ranges of daily electrolyte supply and urinary output5. Even for medical experts, fluid volume assessment, which essentially means determining if an individual might be fluid overloaded or dehydrated, is a difficult task, particularly in the presence of disease7.Interfering with the human fluid volume status by administering an intravenous (iv) infusion solution routinely concerns any medical discipline today10 and aids fluid volume assessment by providing numerical values for total body volume, intracellular and extracellular volume, as well as for fluid load , based on a three-compartment physiologic tissue model8. In our previous non-interventional, observational cohort study on 71 females undergoing gynaecological procedures, we have analysed the results of blinded BCM-measurements before and after general anaesthesia11. Perioperatively, we observed a significant increase in fluid load, extracellular volume and total body volume, amounting to roughly half of the routinely administered infusion solution11.Whole-body bioimpedance spectroscopy using the Body Composition Monitor (BCM) has been extensively validated12 and local inflammation13 resulting in capillary leak14 may influence the perioperative fluid distribution. However, as all subjects in our prior cohort had normal renal function, we were surprised to detect a clinically meaningful fluid shift at all11. A comprehensive search of the literature revealed that the immediate and intermediate effects of intravenous infusion therapy on the fluid compartments of the human body, as well as the fluid volume ‘status quo’ before the start of any intravenous therapy, are not routinely captured in medical practice. Although fluid therapy is a common medical task15, its basic physiological consequences may thus be largely unknown to medical care takers.Various elements like the systemic stress responseBioimpedance spectroscopy can easily be put into practice. The present cross-over study in healthy volunteers was designed to test the efficacy of whole-body bioimpedance spectroscopy for assessment of fluid volume status at baseline, as well as for detection of rapid and longer-term changes in fluid load, extracellular volume, intracellular volume and total body volume (if any), under standardized intravenous fluid therapy. As a secondary objective, we aimed at recording the potential changes in hemodynamic parameters: blood pressure, peripheral capillary oxygen saturation and saturated and heart rate.2, normal renal function and normal hepatic function, and absence of other laboratory value abnormalities , with body mass index 23.1 ± 3.0 kg/mIn study group “Fluid”, fluid load increased from the baseline value of −0.2 ± 1.0 l to the maximal value of 1.0 ± 0.9 l in minute 70 and remained continuously elevated in comparison to baseline throughout minute 300. In study group “Zero”, fluid load decreased from the baseline value of 0.5 ± 1.1 l in minute 0 to the minimal value of −1.1 ± 1.1 l in minute 300 Fig. , Table 2In study group “Fluid”, intracellular volume decreased from the baseline value of 26.8 ± 3.9 l to the minimal value of 26.0 ± 3.9 l in minute 70; intracellular volume thereafter increased, but remained continuously decreased in comparison to baseline throughout minute 300 except minute 230 and 240 Fig. , Table 2In study group “Fluid”, total body volume increased from the baseline value of 45.3 ± 6.3 l to the maximal value of 45.8 ± 6.2 l in minute 230 and remained continuously elevated in comparison to baseline throughout minute 300 Fig. , Table 2Results from the mixed model analysis for AUC of the five studied BCM-derived variables are listed in Table In study group “Fluid”, total urinary volume excretion amounted to 1,897 ± 513 ml by minute 300, while in study group “Zero”, total urinary volume excretion amounted to 679 ± 360 ml Fig. , Table 4Peripheral capillary oxygen saturation, heart rate and blood pressure were measured during the first 90 minutes of the study, as well as at discharge in minute 300 Fig. , Table 5In the present cross-over study, we rapidly infused an “isotonic”, balanced infusion solution into healthy volunteers on study day “Fluid”, and used biompedance spectroscopy to assess their fluid volume changes. After the administration of 2.2 l ELO-MEL-isoton on average within one hour, the fluid volume status underwent clinically meaningful alterations over the short-time infusion period, which remained detectable through the 5-hour observational period. While fluid load, extracellular volume and total body volume increased during the infusion and remained significantly elevated throughout the observational period, compared to baseline and study day “Zero”, intracellular volume decreased during the infusion period and slowly returned to baseline throughout the observational period. Total urinary volume averaged to 1.9 l versus 0.7 l by 5 hours in the “Fluid” versus “Zero” group, respectively, and systolic blood pressure was significantly higher in “Fluid” than in “Zero” between minute 50 and minute 90, when extracellular volume expansion in the “Fluid” group was highest.15, but also appears to be indispensable in severe medical conditions, although the question how to perform adequate fluid resuscitation is perhaps among the ‘hottest topics’ in intensive care medicine. A landmark randomized controlled clinical trial16 indicated that protocolized delivery of early, goal-directed therapy (EGDT) to patients presenting to the emergency department with septic shock reduced hospital mortality and hospital stay. EGDT was aimed at achieving a central venous pressure (CVP) of 8–12 mmHg, and based on this study16, a CVP target of 8–12 mmHg entered the surviving sepsis guidelines17, despite three multicentre trials  after the initial study16 showing that EGDT was not superior to usual care. In another study on fluid resuscitation in septic shock, where a positive fluid balance and elevated CVP were associated with increased mortality21, the authors emphasized the difficulty of stopping the volume infusion, which was not part of the EGDT protocol, and even more drastic terms have been used to describe the dilemma of overloading patients with fluid22.Intravenous fluid therapy is not only common23, while a much earlier, but very small retrospective study showed that at least one day of negative fluid balance achieved by the third day of treatment may be a good independent predictor of survival in patients with septic shock24. A recent randomized, parallel group multicentre feasibility trial in patients with septic shock pointed towards benefit with fluid restriction25. In critically ill patients with acute kidney injury, fluid overload was independently associated with mortality26. Clearly, for acute lung injury, a positive fluid balance has been shown to increase the time spent on mechanical ventilation and to trend toward increased mortality27, while a negative cumulative fluid balance at day 4 was associated with significantly lower mortality28. In surgical patients, a restricted intravenous fluid regimen significantly reduced postoperative complications29, and vice-versa, a positive fluid balance was associated with complications after elective open infrarenal abdominal aortic aneurysm repair30.The potential danger of a positive fluid balance has been emphasized in a large pan-European study of critically ill patients, where a positive fluid balance was among the strongest prognostic factors for death40, how does the present analysis relate? Intravenous fluid therapy may lead to hospital-acquired generalized interstitial oedema (HAGIE)41, but repeatedly, the argument has been brought forth that this is in sick patients with increased vascular permeability, and possibly kidney or heart disease. The fact that an ‘isotonic solution’, infused into healthy males with a normal capillary leak index, leads to a decrease in intracellular volume and fluid accumulation in the extracellular volume, might lead to the conclusion that the infusion itself is responsible for fluid load in the hospital-setting. Importantly however, our healthy volunteers are not an appropriate model for fluid therapy of patients with capillary leak and intracellular derangements form organelle dysfunction, and our results do not imply that we know what the corollary of intracellular fluid shifts and an increase in extracellular fluid really might be.While the list of studies reporting a negative impact of fluid overload in the intensive care setting and elsewhere might be continued unceasingly42. In the past, colloid solutions have been advocated, which, in theory, maintain an effective colloid osmotic pressure or tonicity relative to the extracellular fluid compartment. A systematic (Cochrane) review on colloids versus crystalloids for fluid resuscitation in critically ill patients, however, revealed no evidence that colloids reduce the risk of death43. Among colloids, hydroxyethyl starch is even ineligible for volume resuscitation, as its use in the critically ill is associated with an increased risk of mortality and acute kidney injury44. Adverse outcomes could be mediated by damage done to the fragile, endothelial glycocalyx layer, which is already at risk in patients with sepsis46, undergoes changes through inflammatory and pathological conditions on tissue permeability47, and can be further disrupted by rapid intravenous infusion of fluids49. In view of the present data and the association between a positive fluid balance with adverse outcomes, management of fluid therapy might need to be reassessed.Clearly, the ideal fluid for resuscitation and other purposes has not been identified51, although we acknowledge as previously, that our data from healthy volunteers do not allow a direct jump to this conclusion.In order to rule out that ELO-MEL-isoton was simply the ‘wrong’ infusion solution, while solutions with lower osmolarity might yield different results, a subsequent experiment should be performed with lactated Ringer’s solution (osmolarity = 278 mosmol/l), in comparison with normal saline (osmolarity 308 mosol/l). Alternatively, it might be rewarding to continue searching for a more physiological infusion solution. In addition, treatment with liquids by the oral and gastrointestinal routes of administration should be reconsidered, as in two previous publications52. Patients with cholera, however, suffer from diarrhoea and severe dehydration as a consequence of cholera toxin’s activating cystic fibrosis transmembrane conductance regulator channels in the small intestine, which in turn causes sodium and chloride loss from inside the cells. In contrast to patients with cholera, not all hospitalized patients nowadays are severely dehydrated. It therefore lies at hand that fluid might not necessarily have to be administered intravenously.Studying gastrointestinal fluid administration, however, seems logical as the process of adsorbing fluid and electrolytes through the gut potentially leads to a more adequate intravasal osmolar balance than the administration of an ‘unphysiological’ infusion solution, meanwhile keeping the gut active in patients who suffer from illness, who are otherwise deprived from food and fluid intake during the early hospitalization period, especially in intensive care. In following this rationale, it should be remembered that the origins of intravenous fluid therapy date back to the outbreak of cholera, spreading across Asia and Europe from India where it began in 1829et al. reported a persistent median weight gain of 0.85 kg after infusion of 40 ml/kg lactated Ringer’s solution 24 hours later in healthy volunteers53. Other studies showed that a saline infusion of 22 ml/kg was not excreted until approximately two days later, again in young healthy volunteers55.Returning to the data of the present study, several details merit further discussion. Despite significantly larger urine output after 300 minutes in study group “Fluid”, we also measured a significant weight gain of 0.3 kg at the end of the study day (p = 0.015). This result indicates that fluid actually gets stored, even in healthy subjects. In line, Holte Peripheral oxygen saturation was marginally better in study group “Fluid”, compared to “Zero” in minutes 20, 30 and 70, and systolic blood was higher from minute 50–90, while heart rate was lower at several time-points. These results are in accordance with basic physiological principles. An increase in systolic blood pressure can be explained by the Frank-Starling mechanism, as intravenous fluid therapy increases cardiac filling, which improves cardiac output.Study limitations: The sample size of the present study is small, which represents a study limitation. However, the results of our analysis are likely solid, because the direction of the data points was consistent. Applying a multiple testing procedure would not change the inference from our data analysis: The differences of the area under the curve for fluid load, relative fluid load and extracellular volume between the two groups remain highly significant even after Bonferroni correction for having tested five different outcome variables.56. Whole body bioimpedance spectroscopy seems to be more sensitive in volume changes in the limbs, and therefore, extracellular fluid accumulation in the trunk could be incompletely measured by this method57. Although we cannot formally rule out that bias might have been introduced by the method itself, the BCM had been validated against direct estimation methods in more than 500 healthy subjects10, and is accepted as a very precise whole body bioimpedance spectroscopy device61.We were unable to compare our BCM results with additional reference methods. Using the BCM, bioelectrical properties are measured between wrist and ankle and the three body segments arms, trunk and legs with different cross sectional areas and lengths influence whole body impedance. While the limbs add 90%, the trunk contributes 10% to the impedanceIt has been recommended that subjects should be resting in a supine position for at least 5 minutes, before the BCM-measurement is started, such that fluid volume equilibration has taken place. The spikes in fluid load, relative fluid load and extracellular volume that were observed after the study volunteers rose up to urinate Fig. , c,e mayIn conclusion, when we tested the efficacy of whole-body bioimpedance spectroscopy for assessment of fluid volume status at baseline, as well as for detection of rapid and longer-term changes in fluid load, extracellular volume, extracellular volume, intracelluIar volume and total body volume under standardized, “isotonic” intravenous fluid therapy, we observed a statistically significant, and clinically meaningful decrease in intracellular volume, and an increase in extracellular volume throughout the study period. We posit that the observed fluid volume changes, which occurred in perfectly healthy subjects, may be useful in searching for explanations of the adverse effects that have been reported for fluid therapy in the clinical setting. The present results may moreover inspire our quest to search for a more physiological infusion solution, and to test the gastrointestinal route of fluid administration, even in patients with critical illness.2), absence of hypotension (systolic blood pressure ≤90 mmHg) and hypertension , absence of cardiac insufficiency (NYHA stage ≥2)62 and absence of renal insufficiency (estimated glomerular filtration rate <60 ml/min/1.73 m2 [MDRD-IDMS formula]). Participation in another clinical trial within the last 4 weeks prior to study day 1 precluded the study participation. All participants were recruited from the database of the Medical University of Vienna’s Department of Clinical Pharmacology from October 2014 until March 2016. Initial contact with the participants was established by telephone or mail, to inquire about their interest and availability. Written informed consent was obtained prior to study day 1, and blood was drawn immediately thereafter for analysis of laboratory values at baseline . Capillary leak index (CLI) was calculated as the ratio of CRP (in milligrams per decilitre) over serum albumin (in grams per litre), multiplied by 100, as a parameter for vascular permeability.Study participants had to volunteer, and had to be healthy males, ≥18 years of age. Criteria for health included absence of adiposity  at a rate of 0,50 ml/kg/min  for 60 minutes. The well-validated63 Robinson Formula64 was used for calculation of the ideal body weight31 = /2.54 × 1.9 + 52. In study group “Zero”, participants received no therapy at all, and had to remain thirsting. After completion of study day 1, all participants crossed over into their respective remaining study group, for the second study day  before registration with ClinicalTrials.gov . Randomization into the study group “Fluid” or study group “Zero” was performed on study day 1, using sealed, opaque envelopes. In study group “Fluid”, participants received intravenous fluid therapy with the ELO-MEL-isoton infusion solution  and non-invasive blood pressure (NIBP), as well as by electrocardiogram (ECG), at baseline and in 10-minute intervals thereafter for 90 minutes. BCM-measurements were also performed at baseline and in 10-minute intervals thereafter for 300 minutes, and BCM-electrodes were taped for the whole study day. In the “Fluid” group, a plastic cannula  was inserted into an antecubital vein of the arm contralateral to the BCM-measurement. The infusion solution was started immediately thereafter.On the morning of both study days, participants were admitted to the clinical research ward of the Medical University of Vienna after having fasted 6 hours prior. Participants urinated before determination of the baseline weight. Physical status was recorded using the American Society of Anaesthesiologists’ classification systemAfter the start of the study clock, participants urinated every 60 minutes into a urine receiver, and were reweighed immediately thereafter. The supine position was abandoned during this process. All urine volumes were measured from minute 60 onwards. The entire study duration was 300 minutes on both study days.61. This device was originally designed to determine the fluid volume status including fluid load (termed fluid overload on the BCM-device) in patients with end stage renal disease on renal replacement therapy, thereby aiding the optimal fluid removal during the dialysis session (the ‘dry weight’ prescription)39. Briefly, the BCM determines resistance and reactance at 50 discrete frequencies from 3 to 1,000 kHz. Extracellular and intracellular resistance are obtained on the basis of a Cole model66. Using these read-out values, extracellular volume, intracellular volume, and total body volume are automatically calculated by the BCM-device, employing a fluid model described by Moissl10. Subsequently, excessive extracellular fluid is distinguished from the hydration of major body tissue, on the basis of a physiological tissue model described by Chamney8. The latter model provides the normal hydration status for a human being of given weight, height and sex, i.e. the expected normal values for extracellular volume and intracellular volume that would usually occur in an individual with adequate renal function, and in a state of normohydration. The actual tissue hydration is then calculated from the difference between the normal and the measured values for extracellular volume and intracellular volume. Excess extracellular fluid is provided as ‘fluid overload’ or ‘overhydration’ in litres, and in % above “normal” extracellular volume, as a numerical value on the BCM-device. In dehydrated patients, the value for ‘overhydration’ on the BCM-device reads negative.The BCM is a whole-body bioimpedance spectroscopy device, manufactured and distributed by Fresenius Medical Care (FMC) Germany2), adipose tissue mass, fat tissue index (=adipose tissue mass/height2), and fat mass. Bioimpedance spectroscopy has high reproducibility and sensitivity67. Extensive validation of the fluid volumes and body composition has been performed against reference methods , involving healthy volunteers and haemodialysis patients10.BCM-measurements are painless, requiring the taping of 4 non-recyclable BCM-electrode strips to wrist and ankle, respectively hand and foot, on one side of the body. The results of the BCM-measurements are automatically stored on a patient BCM-card. Besides fluid volume status, a typical BCM-measurement also provides more basic information on the body composition, such as lean tissue mass, lean tissue index , we primarily used the term fluid load and fluid status for our study in healthy volunteers. Similarly, the term extracellular water used in previous publicationsth and 75th percentile [where appropriate]) were used to portray the participants’ characteristics, laboratory values, hemodynamic measures and BCM-results. The primary statistical analysis was performed for the 5 following variables: fluid load, relative fluid load in % extracellular volume, total body volume, extracellular volume and intracellular volume.  These 5 variables were measured every 10 minutes over a period of 300 minutes. Data were systematically missing in minute 200 and in minute 260. For 9 patients, data were missing at random for different time points. Imputation was performed in almost all cases by linear interpolation (taking the mean of the neighbouring data points) except for one patient who had missing data at the final measurement (in minute 300). In the latter case, a simple last observation carried forward strategy was applied and the values from minute 290 were used for imputation. For the imputed data the area under the curve (AUC) was calculated using the trapezoidal rule for all 5 variables. Statistical data analysis for the cross-over trial was performed using for each variable a mixed model (SAS PROC MIXED) with “treatment” and “study day” as fixed effect and “patient” as random effect71. We reported p-values without correcting for multiple testing. Simple Bonferroni correction can be applied in discussing the results if one wanted to take into account that five variables were tested.Descriptive statistics The Statistical Package for Social Sciences (SPSS 20.0) and Microsoft Excel 2010 was used for the analyses mentioned above.All datasets generated during the current study are available from the corresponding author on reasonable request and the results from all data analysed during this study are included in the published article.


Clear cell sarcoma (CCS) is a rare malignant soft-tissue neoplasm that displays melanocytic markers and exhibits striking histopathological features. The tumour has a predilection for the lower extremities and rarely presents in the mediastinum.We present a case of primary mediastinal CCS in a 57-year-old man. Computer tomography (CT) revealed a 12 × 12 × 7.5 cm mass in the anterior mediastinum. Microscopically, the tumour mainly consisted of epithelioid cells with oval vesicular nuclei and eosinophilic cytoplasm. Immunohistochemically, the tumour was positive for human melanoma black 45 (HMB-45) and vimentin but negative for S-100 and Melan-A. Fluorescence in situ hybridisation (FISH) showed a translocation involving the EWSR1 gene region.This report will illustrate that the mediastinum is a potential site for primary CCS and FISH plays an important role in making a conclusive diagnosis. CCS is a rare aggressive sarcoma accounting for only 1% of soft-tissue sarcoma. It usually occurs in adolescents and young adults. The first description of a case of CCS is attributed to Enzinger in 1965, which called it a “malignant melanoma of soft parts” due to its melanocytic differentiation –7.Common tumours in adults that occur in the anterior mediastinum are thymomas, lymphomas, and germ cell tumours. Other soft-tissue tumours that arise in the anterior mediastinum can include leiomyosarcoma, malignant peripheral nerve sheath tumour, liposarcoma, synovial sarcoma, angiosarcoma, and extraskeletal osteosarcoma, among others. A primary CCS in the anterior mediastinum is extremely rare. The present study reports an unusual case of CCS in the mediastinum, confirmed by FISH analysis and immunohistochemical study.A 57-year-old man was admitted to our hospital with complaints of a 1-month history of chest distending pain. He had no history of cutaneous malignancy. On admission, a chest CT revealed a giant lobulated mass with uneven density approximately 12 × 12 × 7.5 cm in size occupying the anterior mediastinum Fig.  and b. TGrossly, the mass was well-demarcated, measuring 10 × 10 × 5 cm in size and yellow-grey on the cut surface. A massive necrosis was identified.Histologically, the tumour demonstrated expansive growth and infiltrated the adjacent lung tissue Fig. . The tumImmunohistochemical studies showed a strong but diffuse distribution of the markers vimentin Fig.  and HMB-Dual colour interphase fluorescence in situ hybridisation utilising the EWSR1 break-apart probe was performed on a paraffin section. A clear separation of red and green signals within a single cell was identified in most cells, which demonstrated the presence of the EWSR1 rearrangement Fig. . No BRAFTo date, 8 months after operation, the patient is well and without evidence of a recurrence or metastasis.CCSs are a distinctive form of soft-tissue neoplasms arising from the deeper soft tissues of the extremities in 90–95% of cases. They make up 1% of all soft-tissue sarcomas and mainly affect young adults aged 20–40, with a slight female bias. CCSs also arise in other rare locations outside of the extremities, such as the kidney , stomachPrimary mediastinal CCSs are extremely rare. Of all published studies on CCSs, there are only 2 reported cases in the literature involving mediastinum , 12. MorThree cases of mediastinal CCSs, including our case, shared some common characteristics, such as a tumour size over 5 cm and a diffuse area of necrosis. Since the mediastinum is the internal space in the thoracic cavity between the lungs surrounded by loose connective tissue, mediastinal tumours tend to grow very large. These tumours produce few symptoms and therefore are often detected at the later stage. With the increase of tumour size, the central area of the tumour is prone to develop necrosis as a result of an insufficient blood supply.Mediastinal CCSs also have similar morphological appearance. As indicated in our case, the tumour was composed of epithelioid cells with ovoid vesicular nuclei and eosinophilic cytoplasm. These cells infiltrated the adjacent lung tissue, showing an aggressive growth pattern. CCS is supposed to derive from neural crest cells and often shows melanocytic differentiation, which is confirmed by the expressions of S-100, HMB-45, and Melan-A. These markers are helpful for distinguishing CCSs from other types of soft tumours. In our case, the tumour cells were positive for HMB-45. However, we were unable to identify either S-100 or Melan-A in multiple sections of the tumour. Due to the inconsistent expression of melanocytic markers and its unusual site, more tumours should be rules out before making a diagnosis of CCS.In patients without cutaneous involvement and a history of melanoma, distinguishing CCS from metastatic malignant melanoma may have diagnostic difficulties because CCS shares striking histological and immunohistochemical similarities with cutaneous melanoma. Fortunately, molecular genetic characterisation of CCS has been shown to be specific for t translocation, which has not yet been identified in melanoma , 14. PreCCS has an aggressive behaviour. Poor prognosis of CCS is closely related to tumour size > 5 cm and the presence of necrosis, metastasis, and local recurrence. Although the patient has not shown any evidence of tumour recurrence or metastasis during the follow-up period for 8 months, the tumour’s diameter > 5 cm and massive necrosis indicated a poor prognosis.In conclusion, we report an extremely rare mediastinal CCS, a high grade of soft-tissue sarcoma with a distinct molecular gene rearrangement and with morphological features. The present case demonstrates that the mediastinum is a possible site of CCS and that a molecular genetics study is necessary for the correct diagnosis.


Microcephaly has become a major public health problem in Brazil. The total number of newborns with microcephaly was reported to be >4000 in June 2016. Studies suggest that Zika Virus is a major cause of new microcephaly cases in Brazil. Inside the uterus, the foetus is surrounded by the Amniotic Fluid, a proximal fluid that contains foetal and maternal cells as well as microorganisms and where Zika Virus was already found.A previous study reported the presence of the Zika Virus in the amniotic fluid  of two pregnant women carrying microcephaly foetuses in Brazil. The virus was detected by means of real-time PCR and metatranscriptomic analysis. We compared the microbiome of these two cases with metatranscriptomic sequences from 16 pregnant women collected at various times in their pregnanciesStreptococcus and Propionibacterium) found in Amniotic Fluid may be involved in neurological diseases. When the foetus is infected by the Zika Virus, due to neurological damage, they do not move inside the uterus, thus changing the Amniotic Fluid environment, potentially leading to secondary problems. Zika infection could also lead to an immunodeficient state, making bacterial colonization of the foetuses easier. An altered microbial composition during pregnancy may also result in harmful secondary metabolite production from certain microbes that further impair foetal brain development. However, these observations of potentially harmful microbial species are correlations and thus cannot be assumed to be causative agents of  disease. In our study, microbial and parasitic diversity of the Amniotic Fluid was lower in patients infected by ZIKV, compared to that of Prenatal and Preterm controls. The present study was a first attempt to shed light on the microbial and parasitic diversity associated with ZIKV-infected pregnant women bearing microcephaly foetuses, and the presence of diverse microbial and parasite communities in the Amniotic Fluid suggests a poor health status of both the pregnant women and the foetuses they carry.Several strains of bacteria  contains supplementary material, which is available to authorized users. Ureaplasma urealyticum, Fusobacterium sp., and Mycoplasma hominis   were also found. Except for Onchocerca, which can cause blindness, none of these parasites appear to have neural tropism   and 1183 (4.8%) reads], Burkholderia [280 (1.47%) and 713 (2.87%) reads], Flavobacterium [244 (1.28%) and 533 (2.15%) reads], Streptococcus [78 (0.41%) and 515 (2.1%) reads], and Paucibacter [146  and 295 (1.19%) reads]  and 625 (31.19%) reads]. In contrast, Ralstonia was the most abundant genus in the Controls [= ~ 2700 (= ~ 20%) reads].ds] Fig. . The fivds] Fig. . EnterobStreptococcus and Propionibacterium) found in AF may be involved in neurological diseases. Amniotic fluid and the placenta contain different types of bacteria, particularly in pre-term birth cases [Clostridium difficile also resulted in a decrease in diversity in the faecal microbiome [p < 0.05) in patients infected by ZIKV, compared to that of Prenatal and Preterm controls (Additional file Propionibacterium was found in women with preterm premature rupture of membranes during pregnancy [Several strains of bacteria (e.g., th cases , 17. A rth cases . Due to th cases . Similarcrobiome . Howevercrobiome , 22. In regnancy . This ge


Intracortical microstimulation (ICMS) is one of the most widely employed techniques for providing causal evidence of the relationship between neuronal activity and specific motor, perceptual, or even cognitive functions. In recent years, several new types of linear multielectrode silicon probes have been developed, allowing researchers to sample neuronal activity at different depths along the same cortical site simultaneously and with high spatial precision. Nevertheless, silicon multielectrode probes have been rarely employed for ICMS studies and, more importantly, it is unknown whether and to what extent they can be used for combined recording and stimulation experiments. Here, we addressed these issues during both acute and chronic conditions. First, we compared the behavioral outcomes of ICMS delivered to the hand region of a monkey's motor cortex with multielectrode silicon probes, commercially available multisite stainless-steel probes and single-tip glass-coated tungsten microelectrodes. The results for all three of the probes were reliable and similar. Furthermore, we tested the impact of long-train ICMS delivered through chronically implanted silicon probes at different time intervals, from 1 to 198 days after ICMS sessions, showing that although the number of recorded neurons decreased over time, in line with previous studies, ICMS did not alter silicon probes' recording capabilities. These findings indicate that in ICMS experiments, the performance of linear multielectrode silicon probes is comparable to that of both single-tip and multielectrode stainless-steel probes, suggesting that the silicon probes can be successfully used for combined recording and stimulation studies in chronic conditions. The electrical stimulation of brain tissue marked the beginning of modern electrophysiology and strongly contributed to neuroscientific progress by allowing researchers to study brain organization and function in many animal species, including humans. Initially, electrical stimulation could be applied only to the cortical surface and induced gross activation that involved large neuronal populations , previously used for other neurophysiological experiments (see below). Before recordings, the monkey was habituated to sitting in a primate chair and to interacting with the experimenters. It was then trained to perform the visuomotor tasks described in previous studies . All experimental protocols were approved by the Veterinarian Animal Care and Use Committee of the University of Parma  and authorized by the Italian Ministry of Health .We performed both acute experiments, in which a single electrode/probe was temporarily inserted into the brain and then retracted at the end of the experimental session, and chronic experiments, in which probes were permanently implanted in the brain tissue for several months.After acute single-cell recording experiments with different types of linear multielectrode probes were conducted . Dexamethasone and prophylactic broad-spectrum antibiotics were administered pre- and postoperatively. Furthermore, analgesics were administered intra- and postoperatively. Dexamethasone administration was continued for 1 week after the surgery. During all surgeries, hydration of the monkey was maintained with continuous infusion of saline solution. A heating pad stabilized the monkey's body temperature throughout the surgical procedure. Heart rate, respiratory depth, and body temperature were continuously monitored. Upon recovery from anesthesia, the animal was returned to its home cage and closely monitored until complete recovery.The neuronal recordings described in this paper were performed by means of two 3D arrays of 64 sputter-deposited platinum electrodes arranged into 2 parallel lines of 4 shafts, each of which had eight recording channels. Probes were implanted with a vertical approach, approximately 1 mm laterally to the mesial wall. Previous reports provide more details on the methodology of probe fabrication , and recorded in sets of 16 channels. All final quantitative analyses were performed offline, as described in the subsequent sections. From the same chronically implanted probes, ICMS was performed at the end of the recording sessions. Monopolar, biphasic trains of cathodic square-wave pulses were delivered through a constant current stimulator , with the following parameters, which are based on previous ICMS studies on area F6  was detected by applying to the filtered  wide-band activity a negative threshold corresponding to 2.5 standard deviations from the mean peak height. Spikes were then sorted using standard principal component and template matching techniques, provided by dedicated offline sorting software (Plexon Inc.), as described elsewhere  relative to those that exhibited the opposite effect (n = 1). Notably, most of the sites that initially showed SUA still did that after ICMS as well (14/16). Figure Figure as well 1/16. FiguFigure In this study, we compared the performance of linear multielectrode silicon probes in acute ICMS experiments in the motor cortex of a monkey with the performance of linear multielectrode stainless-steel probes and classical tip electrodes. In addition, we verified the long-term single-neuron recording capabilities of chronically implanted silicon probes previously used for both ICMS and single-neuron recordings.The results of the acute ICMS experiments showed that all three probes could trigger similar behavioral reactions at comparable current-intensity thresholds. We used a single tip-electrode for comparison because most extant ICMS studies on both motor and nonmotor brain regions have been carried out with this type of electrode. However, because we sequentially replicated cortical penetrations in the same position with different probes , it could be argued that we failed to capture subtle differences among probes, thus producing false-negative results. Nevertheless, it is worth noting that, with all probes, the minimum and average current-intensity thresholds reflect the well-established properties of the motor cortex, that is, greater excitability of the cortical sites located closer to or within the anterior bank of the central sulcus , to deliver ICMS at all the available sites  of microelectrode arrays chronically implanted in 27 monkeys over a period of about 15 years showed that the recording duration ranged from 0 to 2,104 days, with a considerable case-by-case variability and with a number of possible different failure modes (Barrese et al., An important limitation of the present study consists in the low number of observations carried out on a single animal with a single set of probes. A comprehensive retrospective report on a large set (CF performed the experiments, analyzed the data, prepared figures and wrote and revised the manuscript; MM performed the experiments, analyzed the data and revised the manuscript; AL performed the experiments, analyzed the data and revised the manuscript; ML performed the experiments, analyzed the data and revised the manuscript; LB designed the research, supervised the experiments, wrote and revised the manuscript.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.


The increasing popularity of hookah (or waterpipe) use in the United States and elsewhere has consequences for public health because it has similar health risks to that of combustible cigarettes. While hookah use rapidly increases in popularity, social media data  can be used to capture and describe the social and environmental contexts in which individuals use, perceive, discuss, and are marketed this tobacco product. These data may allow people to organically report on their sentiment toward tobacco products like hookah unprimed by a researcher, without instrument bias, and at low costs.This study describes the sentiment of hookah-related posts on Twitter and describes the importance of debiasing Twitter data when attempting to understand attitudes.Hookah-related posts on Twitter  were collected from March 24, 2015, to December 2, 2016. Machine learning models were used to describe sentiment on 20 different emotions and to debias the data so that Twitter posts reflected sentiment of legitimate human users and not of social bots or marketing-oriented accounts that would possibly provide overly positive or overly negative sentiment of hookah.From the analytical sample, 352,116 tweets (59.50%) were classified as positive while 177,537 (30.00%) were classified as negative, and 62,139 (10.50%) neutral. Among all positive tweets, 218,312 (62.00%) were classified as highly positive emotions , while 133,804 (38.00%) positive tweets were classified as passive positive emotions . Among all negative tweets, 95,870 (54.00%) were classified as subdued negative emotions  while the remaining 81,667 (46.00%) negative tweets were classified as highly negative emotions . Sentiment changed drastically when comparing a corpus of tweets with social bots to one without. For example, the probability of any one tweet reflecting joy was 61.30% from the debiased (or bot free) corpus of tweets. In contrast, the probability of any one tweet reflecting joy was 16.40% from the biased corpus.Social media data provide researchers the ability to understand public sentiment and attitudes by listening to what people are saying in their own words. Tobacco control programmers in charge of risk communication may consider targeting individuals posting positive messages about hookah on Twitter or designing messages that amplify the negative sentiments. Posts on Twitter communicating positive sentiment toward hookah could add to the normalization of hookah use and is an area of future research. Findings from this study demonstrated the importance of debiasing data when attempting to understand attitudes from Twitter data. The popularity of hookah (or waterpipe) use is increasing among youth and young adults in the United States and elsewhere . This inIn a recent study analyzing hookah-related posts on Instagram, researchers reported that hookah was often cross-promoted with alcohol by nightclubs, bars, restaurants, and hookah lounges . InstagrIn this study, we demonstrate the feasibility of a Twitter-based “infoveillance”  methodolData were obtained from Twitter’s Streaming Application Program Interface (API) based on Twitter4J libraries, an open source database of java language used to analyze data from the API. Software was written to automate this process. Tweets posted between March 24, 2015, and December 2, 2016, were collected. The root terms used to collect the sample of tweets were hookah(s) or hooka(s) or sheesha(s) or shisha(s) or sesh(s). The root terms could have appeared in the post or in an accompanying hashtag, for example, hookah or #hookah. While the word waterpipe is commonly used in academic papers and presentations to refer to hookah, it is uncommon for individuals to use this term on social media and it was thus not included in this study .To determine the performance of the rule-based reasoning model, the sentiment output of the tweets was analyzed. The rule-based reasoning sentiment analysis provided an The rule-based reasoning model was considered as a reference point, or baseline model, to inform a model based on a support vector machine (SVM) algorithm. Combining a manual rule‒based method with an automated one like SVM provided us with a more generalized solution for sentiment analysis. In the implementation of this algorithm, the model was trained on the SemEval  and the F=0.90).In order to utilize an SVM model, the data have to be prepared as follows. (1) First the tweets are cleaned to permit uniform analysis using a process called tokenization that involves removing uniform resource locators (URLs) and unrecognizable UTF-8 encoding forms from the text of tweets along with stop words such as “the” and “an,” which frequently occur in spoken and written language but do not convey sentiment. Hypertext markup language (HTML) tags were replaced by whitespace, and then a pattern-matching algorithm was used to recognize any trailing URL patterns that remained in the text. (2) This preprocessing step also included allowing the model to recognize emoticons , slang, and aggravated language usage . Emoticons were unified so that all types of smiley faces were considered one, while all types of sad faces were considered one. Slang was corrected and punctuation was fixed, for example, “don’t” and “dont” were treated as one. Gerunds or stemming in NLP were handled by the program Porter’s Stemmer , and eloIn order to quantify and compare the impact of bias from social bots, we reduced the 20 levels of emotions down to 5 primary emotions  as described by Bradley et al . We thenA majority of tweets 352,116 (59.50%) were classified as positive while 177,537 (30.00%) were classified as negative , and 62,Social media data provide researchers the ability to understand public sentiment and attitudes by listening to what people are saying in their own words. By using Twitter data, we had the ability to document and describe individuals’ sentiment toward a tobacco product without being primed by a researcher, allowing for spontaneous comments to emerge. We found that the majority of hookah-related posts on Twitter conveyed positive sentiment. Positive posts on Twitter may serve as a screener indicating that an individual is in need of tobacco-related education to reduce misconceptions about a product or to reduce positive social norms about that product. Tobacco control programmers in charge of risk communication may consider targeting individuals posting positive messages about hookah on Twitter or designing messages that amplify the negative sentiments. Twitter may be used to bolster the reach and delivery of health information that communicates the risk of tobacco use -27, and In comparison to earlier studies that relied on sample sizes small enough to reasonably code by hand ,10 durinWhile in this study, 30% of hookah-related posts contained negative sentiment, earlier studies found that negative posts were relatively rare. For example, Grant et al reported 3% of Twitter posts in their data conveyed negative sentiment . This diIn this study, 296,338 Twitter posts were removed because they were found to be from social bots and would not accurately reflect individual sentiment toward hookah use. This number reflects 30% of the initial data that comprised the corpus of tweets. Removing social bots from the analytical corpus had marked effects on the results. The bot free corpus reflected that the overall sentiment was positive . The corpus that included bots and human accounts together reflected that only 33.9% of tweets were positive. While sentiment in tweets is only one of many possible ways bias from social bots can distort Twitter data, the findings from this study highlight the importance of debiasing data collected from Twitter in order to uncover the rich and nuanced information available to health researchers relying on social media data and aimed at understanding public attitudes .Data relied on Twitter’s Streaming API, which prevented us from collecting tweets from private Twitter accounts. As a result, findings may not represent the sentiment toward hookah from individuals with private accounts. The result that more tweets were found to convey a positive sentiment than a negative one should be considered circumstantial and not absolute. While we identified the overall sentiment of posts, we did not track individual users to see whether their posts about hookah change over time. Additionally, a post classified as negative does not necessarily mean the person dislikes hookah. Rather, it means the post had more words conveying negative sentiment than positive. The method used in this study for bot detection is not a perfect system but scores a detection accuracy above 95% suggesting biases from inappropriate removal of legitimate accounts is minimal .Despite these limitations, this study demonstrated the utility in using social media data to understand public attitudes that may influence acceptance of tobacco products such as hookah and defined types of positive and negative attitudes that could be incorporated into public health media campaigns to reduce acceptance of hookah. It also illustrated the importance of debiasing Twitter data so that posts reflect sentiment of legitimate human users and not of social bots or marketing-oriented accounts that could possibly provide overly positive or overly negative sentiment of hookah. Findings should spur efforts to better understand the consequences of hookah-related discussions on Twitter as an informative tool in planning tobacco control efforts.


Patiria pectinifera (phylum Echinodermata). Furthermore, analysis of transcriptome sequence data from the starfish Asterias rubens revealed two PP/OK‐type precursors: an SMP‐type precursor  and a second precursor (ArPPLNP2). We reported previously a detailed analysis of ArPPLNP1 expression in A. rubens and here we report the first functional characterization ArPPLNP2‐derived neuropeptides. Sequencing of a cDNA encoding ArPPLNP2 revealed that it comprises eleven related neuropeptides (ArPPLN2a‐k), the structures of several of which were confirmed using mass spectrometry. Analysis of the expression of ArPPLNP2 and neuropeptides derived from this precursor using mRNA in situ hybridization and immunohistochemistry revealed a widespread distribution, including expression in radial nerve cords, circumoral nerve ring, digestive system, tube feet and innervation of interossicular muscles. In vitro pharmacology revealed that the ArPPLNP2‐derived neuropeptide ArPPLN2h has no effect on the contractility of tube feet or the body wall‐associated apical muscle, contrasting with the relaxing effect of ArPPLN1b (ArSMP) on these preparations. ArPPLN2h does, however, cause dose‐dependent relaxation of cardiac stomach preparations, with greater potency/efficacy than ArPPLN1b and with similar potency/efficacy to the SALMFamide neuropeptide S2. In conclusion, there are similarities in the expression patterns of ArPPLNP1 and ArPPLNP2 but our data also indicate specialization in the roles of neuropeptides derived from these two PP/OK‐type precursors in starfish.Molluscan pedal peptides (PPs) and arthropod orcokinins (OKs) are prototypes of a family of neuropeptides that have been identified in several phyla. Recently, starfish myorelaxant peptide (SMP) was identified as a PP/OK‐type neuropeptide in the starfish  Thus, the evolutionary origin of PP/OK‐type neuropeptides can be traced to the bilaterian common ancestor of protostomes and deuterostomes Jekely, . HoweverAplysia californica on account of its expression by neurons in the pedal ganglia  that encode precursors of PP‐like neuropeptides  provided the first evidence that PP/OK‐type peptides also occur in deuterostomes  was identified as a PP/OK‐type neuropeptide  and we have named the other A. rubens PP/OK‐type precursor A. rubens PP‐like neuropeptide precursor 2 (ArPPLNP2).A homolog of the A. rubens using mRNA in situ hybridization and immunohistochemistry  with a diameter >4 cm were collected at low tide from the Thanet coast  or were obtained from a fisherman based at Whitstable . These animals were maintained in a circulating seawater aquarium at ∼12°C in the School of Biological and Chemical Sciences at Queen Mary University of London and were fed on mussels (Mytilus edulis). Smaller juvenile specimens of A. rubens (diameter 0.5–1.5 cm) were collected at the University of Gothenberg Sven Lovén Centre for Marine Infrastructure  and fixed in Bouin's solution.Starfish  of PP‐like neuropeptide precursor 2 (ArPPLNP2), which comprises a 31‐residue N‐terminal signal peptide and seven putative neuropeptides bounded by dibasic cleavage sites  and a contig (GAUS01023767.1) comprising the 3′ region of PPLNP2 was identified in Asterias forbesi. Combining the partial PPLNP2 transcript sequences from A. rubens and A. forbesi, forward and reverse primers  were designed to enable PCR amplification of a full‐length ArPPLNP2 cDNA from A. rubens.Analysis of The methods used for RNA extraction, cloning and sequencing were as described previously  visualization of ArPPLNP2 transcripts in sections of A. rubens were the same as reported previously in a study investigating expression of the relaxin‐like gonad‐stimulating peptide precursor in A. rubens (AruRGPP)  production of anti‐sense and sense DIG‐labeled RNA probes, (b) preparation of sections of fixed specimens (diameter 4–6 cm) of 2.5The peptide ArPPLN2h (GRTSLSGSSGLTHLSSGFH) was selected as a target antigen for production of rabbit antibodies because it is the most abundant of the ArPPLN2‐derived neuropeptides, occurring in triplicate whilst other peptides occur singly in the precursor. Furthermore, ArPPLN2h shares between 58 and 95% sequence identity with the other ArPPLNP2‐derived neuropeptides. A peptide comprising the C‐terminal octapeptide of ArPPLN2h but with the addition of N‐terminal lysine and tyrosine residues (KYTHLSSGFH) was designed as an antigen (ag) peptide (ArPPLN2h‐ag) and synthesized by Peptide Protein Research Ltd. . Rabbit immunization with the ArPPLN2h‐ag–thyroglobulin conjugate and serum collection was performed by Charles River Labs , as described previously for ArPPLN1b , employing the same methods as described previously for ArPPLN1b  visualization of ArPPLN2h expression in sections of A. rubens using immunohistochemistry (including pre‐absorption tests) and (c) photography of immunostained sections and preparation of montages were the same as those reported previously for ArPPLN1b  preparation of sections of fixed specimens (diameter 4–6 cm) of 2.7A. rubens, using the same methods reported previously for ArPPLNP1b , which is a known relaxant of the cardiac stomach, apical muscle, and tube feet in A. rubens  with 100 µL of 1 mM ArPPLN2h.Consistent with the selection of ArPPLN2h as a representative neuropeptide for generation of antibodies (see above), ArPPLN2h was also selected as a representative ArPPLNP2‐derived peptide for in vitro and in vivo pharmacological experiments. ArPPLN2h was synthesized by Peptide Protein Research Ltd.  and was tested on in vitro preparations of the cardiac stomach, apical muscle and tube foot from 33.15 located in the N‐terminal region and Leu14 and Phe18 located in the C‐terminal region . The ArPPLNP2 ORF encodes a 563 amino acid residue protein comprising a 31‐residue signal peptide and 13 copies of predicted PP/OK‐type peptides, which we have named ArPPLN2a‐k according to their position in the precursor protein. There are three copies of ArPPLN2h and one copy of each of the other peptides  on three in vitro preparations from A. rubens: apical muscle, tube feet and cardiac stomach. No effects on the contractile state of apical muscle and tube foot preparations were observed, whereas ArPPLN2h caused relaxation of cardiac stomach preparations. ArPPLN2h caused dose‐dependent relaxation of cardiac stomach preparations and comparison of the effects of ArPPLN2h with the SALMFamide neuropeptide S2 revealed that the potency and efficacy of ArPPLN2h was very similar to that of S2  observed at 10−6 M was 40.05 ± 5.07% and 48.03 ± 8.17% reversal of KCl‐induced contraction for ArPPLN2h and S2, respectively.ArPPLN2h was tested at a high concentration . ArPPLNP2 comprises 13 copies of 11 PP/OK‐type peptides (ArPPLN2a‐k), with three copies of ArPPLN2h and one copy each of the other ten peptides. Alignment of ArPPLNP2‐derived peptides with other PP/OK‐type neuropeptides reveals several conserved structural characteristics , although it is thought to comprise sensory neurons, interneurons and motor neurons   and in A. planci  suggests that both of these PP/OK‐type precursors would have been present in the common ancestor of all extant starfish. Previous studies have shown that PPLN1‐type (SMP‐type) peptides act as muscle relaxants in P. pectinifera  and therefore it seems likely, based on asteroid phylogeny . It will be interesting, therefore, to investigate if PPLN2‐type peptides also act as muscle relaxants in starfish that belong to other starfish orders—for example, in P. pectinifera and/or A. planci, both of which are species belonging to the order Valvatida. Looking beyond starfish, it would be of interest to determine if PP/OK‐type neuropeptides act as muscle relaxants in other echinoderms Addressing this issue is feasible because precursors of PP/OK‐type neuropeptides have been identified in other echinoderms, including two precursors in the sea urchin S. purpuratus (SpPPLNP1 and SpPPLNP2; Rowe & Elphick, A. japonicus (Rowe, Achhala, & Elphick, This study of PPLN2‐type peptides in As discussed in detail previously (Lin, Egertová et al., The authors declare that they have no conflict of interest.


Colorectal cancer is one of the most common forms of cancer. Spread of tumour to the peritoneal cavity may lead to seeding of cancer cells that adhere to and invade the peritoneal membrane causing peritoneal carcinomatosis. Matrix metalloproteinases (MMPs) play an essential role in cancer cell invasion and dissemination. The aim of this study was to evaluate the morphology and presence of matrix metalloproteinases in peritoneal carcinomatosis. Biopsy samples of the parietal peritoneum were taken from patients undergoing cytoreductive surgery for peritoneal carcinomatosis. The samples were fixed in formalin, dehydrated and embedded in paraffin prior to cutting into 4-µm slices. Staining with haematoxylin/eosin was used for morphology studies, and MMP-1, MMP-2 and TIMP-1 levels were evaluated using immunohistochemistry and light microscopy. The microscopically tumour-free areas of the peritoneal membrane were thin compared to the peripheral invasion zone and the areas invaded by tumour. Peritoneum invaded by tumour was richly vascularised and contained inflammatory cells. MMP-1 was expressed in tumour-free peritoneum and in the invasion zone between tumour and peritoneal tissue, but not in tumour-invaded areas. MMP-2 and TIMP-1 were mostly expressed in the proximity of blood vessels and inflammatory cells in tumour-invaded areas, but was not seen in tumour-free areas. MMPs play an important role in the process of cancer cell invasion of the peritoneum in peritoneal carcinomatosis. The peripheral zone of the tumour appears to be of importance for tumour invasion. It ct person . Earliert person , 5, whilt person . PC has t person , 8. HoweThe cancer invasion process is complex and involves the degradation of the peritoneal basal membrane. Proteolytic enzymes such as zink- and calcium-dependent matrix metalloproteinases (MMPs), produced by both tumour cells and the surrounding stroma, play an important role in this process . One funMMP-1 is a collagenase that degrades the extracellular matrix (ECM) of the interstitial stroma, in particular collagen Types I, II and III. In CRC, increased MMP-1 expression correlates with advanced colon cancer stage and poor prognosis .MMP-2 and MMP-9 belong to the gelatinase subfamily of MMPs, and even though the main substrates for these enzymes are collagen Type IV and gelatin, they share proteolytic activity against other ECM molecules as well. Many studies on CRC have revealed a correlation between increased MMP-2 and MMP-9 expression and poor outcome. Even increased MMP-2 expression in plasma has been shown to be associated with lymph node metastasis in patients with CRC compared to those without .The invasion process is multifactorial and includes factors such as inflammation, the magnitude of surgical trauma, and the presence of ECM-degrading proteases such as matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) Fig. a. HoweveThe aim of this study was to investigate matrix metalloproteinase expression in biopsy samples taken from patients with peritoneal carcinomatosis of colorectal origin.During 2015, three patients undergoing total peritoneal resection by cytoreductive surgery (CRS) where included in the study after having given informed consent. The CRS procedure includes the resection of all or a major part of the parietal peritoneal membrane, with large amounts of tissue being removed. For the purposes of this study, smaller tissue samples (50 mm × 50 mm) where gently dissected away in the operation theatre. Care was taken not to damage the surface and to place the sample with the mesothelial side upwards. Biopsy samples were taken from areas with peritoneal carcinomatosis including the peripheral zone with invading cancer cells, as well as macroscopically tumour-free peritoneal tissue  during fixation Fig. c. TissueFormalin-fixed and paraffin-embedded 4-µm tissue slices were mounted on adhesion slides , deparaffinised, rehydrated in xylene/alcohol gradients (99.5–70%) and rinsed twice in 5 mM Tris-buffered saline solution (TBS), pH 7.8 . The preparations were heated for 15 min at 95 °C in 0.01 mol/L citrate buffer, pH 6.1 as target retrieval solution . Following rinsing with TBS, the preparation slides were mounted on Shandon coverplates . Non-specific protein binding was initially done using 5% fat-free dry milk in TBS, followed by endogenous peroxidase blockade using parts from the Dual Link System-HRP kit .Monoclonal mouse anti-human MMP-1 was diluted to 1 µg/mL , MMP-2 diluted to 2 µg/mL  and TIMP-1 diluted to 4 µg/mL  in 5% fat-free dry milk in TBS. As a negative control, monoclonal mouse antibodies of isotope IgG , the specificity of which is directed towards an enzyme that is neither present nor inducible in mammalian tissues was used.Following incubation with peroxidase labelled polymer conjugated to goat anti-mouse immunoglobulin, the sections were incubated using 3,3′-diaminobenzidine (DAB+) as a chromogenic substrate generating a brownish colour indicating a positive signal, all according to the manufacturer’s instructions .After rinsing with TBS, counterstaining was performed using Harris HTX and mounting achieved with Pertex  following dehydration using alcohol (70–99.5%) and xylene as previously described. Similar techniques have been described previously , 15.Immunohistochemical evaluation was performed by photographic documentation using the Eclipse E800 microscope connected to CoolPix 995 photographic equipment . Immunohistochemical staining was performed in several serial dilutions using duplicates for each dilution.Histologically, the macroscopically tumour-free peritoneal tissue differed from tissue invaded by cancer cells in several respects. The peritoneum in close proximity to the tumour was thicker and had more vessels and inflammatory cells than the microscopically tumour-free areas of the peritoneal membrane. Peritoneal tissue invaded by cancer had a more rigid and fibrotic appearance in the mesothelial and submesothelial surface, compared to tumour-free peritoneum Fig. .Fig. 2MiA thin layer of mesothelial cells was seen covering not only the tumour-free peritoneum, but also areas of peritoneum invaded by tumour Fig. e–f.Morphological evaluation of micrographs of the Htx/Eos stained slices showed inflammatory cells and rich vascularisation in the peritoneal areas invaded by cancer cells and in the immediate adjacent area. This is in contrast to the tumour-free peritoneal membrane where only limited signs of inflammation were seen Fig. .Fig. 3MiMatrix metalloproteinase 1 (MMP-1) expression in tumour-invaded peritoneum was more intense in the peripheral invasion zone between the tumour and normal peritoneal tissue, indicating an active role of MMP-1. In the microscopically tumour-free areas, a more general expression of MMP-1 was seen Fig. .Fig. 4MaMatrix metalloproteinase-2 (MMP-2) expression in the tumour-invaded areas was only seen in the proximity of inflammatory cells and blood vessels Fig. . No exprTissue inhibitor of metalloproteinase-1 (TIMP-1) expression in peritoneum invaded by tumour was only sparsely seen in the invasion zone between the tumour and normal peritoneal tissue and close to the inflammatory cells present Fig. . No exprPeritoneum covers the inside of the abdominal wall as well as the internal organs. A monolayer of mesothelial cells lining the basal membrane of the peritoneum , 2. WhenThe present study investigated the morphology as well as MMP and TIMP-1 levels in normal peritoneal tissue and peritoneum invaded by cancer cells, in samples taken during CRS for peritoneal carcinomatosis.We demonstrated that in peritoneal carcinomatosis, microscopically tumour-free peritoneal tissue differs from peritoneal tissue invaded by tumour cells. The peritoneum of tumour-free areas was thinner than the peripheral zone of the tumour-invaded tissue and the areas invaded by tumour. The peritoneal membrane is a highly dynamic organ and is able to adapt in both structure and function . It has We were also able to demonstrate the presence of inflammatory cells in the region where tumour had invaded the peritoneal membrane. Such inflammatory cells were not present to the same extent in the tumour-free areas Fig. . The estWe observed the presence of a thin layer of mesothelial cells lining not only microscopically healthy peritoneum but also peritoneum invaded by cancer. The function of a mesothelial layer lining tumour tissue is not understood. Signal transmission pathways between the peritoneal mesothelial cells and the invading tumour are complex and the mesothelial layer could have several roles.One of these roles is that the mesothelial cells of the peritoneal membrane are unique in their ability to repair damage following trauma or surgery, as demonstrated by Mutsaers and coworkers . The perDuring normal and pathological conditions, mesothelial cells have the capacity to promote the formation and deposition of fibrin as part of the peritoneal repair process. Not only do mesothelial cells build extracellular matrix, but they also have the capacity to promote its degradation. By producing plasminogen activators and their inhibitors, mesothelial cells are important in the conversion of plasminogen to plasmin causing local fibrin degradation and a general regeneration in tissue repair . Matrix The roles played by MMPs and TIMPs in PC are not fully understood. Desmeules et al.  investigIn the present study, the presence of MMP-2 in tumour-invaded peritoneum was only seen in the proximity of inflammatory cells and vascular structures. No expression of MMP-2 was seen close to the tumour-invaded area or in tumour-free parts of the peritoneal tissue. The presence of TIMP-1 was only seen in the zone between tumour and peritoneal tissue and in the proximity of inflammatory cells. No expression could be seen in microscopically tumour-free peritoneal tissue samples.The role of MMP-1 has previously been discussed in relation to splenic angiosarcoma, a cancer that can metastasise to the membranes of both the peritoneum and pleura, much like mesothelioma. Using immunohistochemical staining, the authors observed that the tumour expressed MMP-1, an enzyme known to increase the invasiveness of mesothelioma cells. The authors suggested that MMP-1 generates a fibrous stroma that aids invasion of the tumour . In the The process of tumour invasion in PC is complex and involves the degradation of the peritoneal basement membrane. Proteolytic enzymes produced by stromal, mesothelial and tumour cells play an important role in this inflammatory process, which depends, among other things, on the magnitude of surgical trauma and the presence of extracellular matrix degrading proteases such as MMPs and their inhibitors (TIMPs), although our knowledge about these biological interactions is limited.The present work adds to our knowledge regarding the presence of increased levels of MMP-1 in PC. Peritoneal carcinomatosis involves a complicated sequence of interdependent stages, from tumour cells released from a primary tumour in the abdominal cavity that might be affected by intraoperative trauma during surgery, via ongoing inflammation and other factors, to the unique capacity of the tumour cells to invade the peritoneum. Proteases seem to play an important role in the invasion process.The study provides increased knowledge of the role of proteases in peritoneal carcinomatosis where MMP-1 appears to be of importance for cells invasion of the peritoneum. Further research investigating the components of the peritoneal metastatic cascade is needed to develop more successful therapeutic strategies for this mortal condition.


Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore, rapid, cost-effective and reliable detection of mutations is an object of extensive research. Today, Förster resonance energy transfer (FRET) probes are among the most often used tools for the detection of nucleic acids and in particular, for the detection of mutations. However, multiple parameters must be taken into account in order to create efficient FRET probes that are sensitive to nucleic acid mutations. In this review; we focus on the design principles for such probes and available computational methods that allow for their rational design. Applications of advanced, rationally designed FRET probes range from new insights into cellular heterogeneity to gaining new knowledge of nucleic acid structures directly in living cells. Nucleic acids are essential for all known forms of life. In the form of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), they function in encoding, transmitting and expressing genetic information . To ensuUpon hybridization, two complementary nucleic strands bind to each other, forming a duplex. Currently, nucleic acid hybridization is seen as a thermodynamically driven process which is promoted by hydration forces and stacking interactions between nucleobases . A practEstablishing design strategies is crucial for oligonucleotide probes that will target and sense nucleic acids and, in particular, single-nucleotide polymorphisms (SNPs) or, in other words, mutations. So far, diverse parameters of modified oligonucleotides have been addressed separately. This includes binding affinity assessments by thermodynamic models , SNP detFluorescence is a convenient method of nucleic acid detection . Among oIn this review we describe recent advances in the design of effective FRET probes. Herein we mainly focus on probes that detect clinically and biologically actionable nucleic acid mutations. Many FRET probes and assays described in the literature potentially can be expanded to the detection of SNP although this is not described in original papers. Herein we introduce such examples as well. An important and novel aspect of this review is the description of recent advances of molecular modeling and quantum chemical approaches for design of effective FRET probes. As demonstrated below, synergetic application of advanced computational chemistry with the chemistry of modified oligonucleotides and biophysics can provide breakthrough scientific and diagnostic settings addressing challenging biological and biomedical tasks.E) of FRET depends on two parameters, the distance, r, between the acceptor and the donor, and the Förster distance, R0. This can be expressed as follows . I. I104]. 11 (Fluorescent nucleobase analogues are other possible FRET donors and acceptors . Even th11 b 105]. . 11 (10512, List et al  studied Mutation-specific probes which use FRET have been successfully applied in the life sciences since the 1990s. Over this period, multiple probe designs have been developed. In general, single-stranded linear, hairpin-shaped or double-stranded probes are most often applied. Among these, hairpin-shaped probes represent one of the most popular designs for SNP detection in vitro and in vivo. However, multiple factors affect the performance of FRET probes in vitro and especially in vivo. These include, but are not limited to, the positioning, number and chemistry of fluorophores, as well as the incorporation of specific recognition and DNA enzyme sequences, all components used in the construction of new probes.Chemical modification of DNA and RNA probe backbones improves specificity of SNP recognition. In particular, LNA has a profound effect on the affinity and, most importantly, specificity of target recognition.An important aspect of FRET, making it an attractive technology in cellular research, is its ability to detect and monitor many of the reactions in live cells. It makes this approach a valuable tool for future in vivo visualization of cellular processes. This direction is rapidly developing and is putting new demands on the rational design of FRET probes. Another important application of FRET probes is the real-time monitoring of DNA amplification in PCR. Utilization of FRET permits the simultaneous detection of multiple products and is highly suitable for clinical diagnostic applications.The use of computational strategies is a rapidly developing research area which carries great promise for mutation-sensitive FRET probes. This novel direction in probe design develops our fundamental understanding of fluorophores’ interaction with each other and with nucleic acids. Besides a deeper knowledge of nucleic acid structure, optical properties and dynamics, computational strategies lead to the successful design of successful probes in silico, i.e., prior to synthesis. The resulting probes are not restrained by the limitations inherent to existing rational design principles but instead apply fundamental knowledge of biomolecular interactions in order to gain more versatile energy transfer and, hence, excellent target detection.


Alterations in integrin expression and function promote tumour growth, invasion, metastasis and neoangiogenesis. Head and neck cancers are highly vascular tumours with a tendency to metastasise. They express a wide range of integrin receptors. Expression of the αv and β1 subunits has been explored relatively extensively and linked to tumour progression and metastasis. Individual receptors αvβ3 and αvβ5 have proved popular targets for diagnostic and therapeutic agents but lesser studied receptors, such as αvβ6, αvβ8, and β1 subfamily members, also show promise. This review presents the current knowledge of integrin expression and function in squamous cell carcinoma of the head and neck (HNSCC), with a particular focus on the arginine-glycine-aspartate (RGD)-binding integrins, in order to highlight the potential of integrins as targets for personalised tumour-specific identification and therapy. Head and neck cancer is a term used for any malignancies that originate from the oral cavity, oesophagus, pharynx, larynx, paranasal sinuses and nasal cavity . The mosSurgery is the mainstay of HNSCC treatment, but many patients present with locally advanced disease (characterized by lymph node metastasis) which requires additional radiotherapy and/or chemotherapy . SurgicaIntegrins are heterodimeric transmembrane glycoproteins consisting of an α-subunit and a β subunit . In vertA number of the RGD-recognising integrins, αvβ3, αvβ5, αvβ8 and α5β1 are involved in controlling angiogenesis ,24,25. αThe biological potential and possible approaches to integrin targeting in general have been reviewed by many researchers ,39,40,41Investigation of changes in integrin expression on tumour tissues identifies possible biomarkers for disease progression, and targets for imaging and drug delivery agents. Additionally, linking integrin expression or signalling to tumour growth, dissemination, or response to therapy identifies areas where targeted integrin inhibitors may improve prognosis The expression of RGD-binding integrins in clinical tissue samples is summarized in The αv subunit is present in a number of integrins involved in neoangiogenesis, migration and invasion. Associated with these processes is activation of matrix metalloproteinases (MMPs), and activation of TGF-β. αvβ3 and αvβ5 have been the focus of significant research as anti-angiogenic targets ,49,50, bThe αv integrin subunit is overexpressed in oesophageal, orbital, eyelid and periorbital squamous cell carcinomas (SCCs), and its expression is associated with tumour invasion and metastasis . High exαv expression has been shown to be higher in tissues from radioresistant nasopharyngeal carcinomas than in tissues from patients who responded to radiotherapy. In vitro, αv expression in the human nasopharyngeal cell line CNE-2 is upregulated in multi-cellular spheroids compared to standard culture conditions, and blocking αv integrin signaling in the SAPK/JNK pathway increased the radiosensitivity of multi-cellular spheroids in vitro and in vivo . DownregOverall, αv expression appears useful as a biomarker of HNSCC tumour tissue and is amenable for biological targeting as an anticancer strategy. The development of small molecule drugs against αv subfamily integrins is feasible, but targeting a particular subfamily member has so far been a more attractive strategy to the pharmaceutical industry . This reαvβ3 and αvβ5 are the most thoroughly studied of the αv subfamily. αvβ5 integrin expression has been found to be significantly higher in oral tumour cells and tumour stroma than in control samples of normal oral cavity squamous epithelium , and sigIn contrast, Jones et al. demonstrated that αvβ5 integrin is expressed in both normal and tumour cells of oral squamous cell carcinoma and the expression of αvβ5 was weak or absent in poorly differentiated lesions . In vitrIntegrin αvβ3 is strongly implicated in tumour angiogenesis . αvβ3 inMultiple preclinical studies have shown that dual αvβ3 and αvβ5 antagonists inhibit tumour angiogenesis and tumour growth . The proIn a single patient case report, cilengitide with gemcitabine inhibited the growth of a highly vascularized head and neck tumour and prolonged life for more than one year, encouraging further investigation in HNSCC  despite Subsequently Heiduschka et al. reported that cilengitide in combination with cisplatin increased apoptosis and gave synergistic growth inhibition in three HNSCC cell lines . Cilengitide in combination with irradiation gave significant inhibition of colony formation . In ex vThe trial investigators suggested that the failure of cilengitide in head and neck cancer is because HNSCC cells, in contrast to tumour endothelium, tend to express low levels of αvβ3, the integrin most tightly bound by cilengitide . αvβ5 inMultiple studies have found αvβ6 is overexpressed in HNSCC. One study reported overall that 95% of HNSCCs express this integrin on tumour cells, whereas αvβ3 and αvβ5 are found on tumour-associated vasculature and stromal cells, respectively . αvβ6 exIn in vitro studies, αvβ6 appears to be important for cell migration. Inducing expression of the β6 subunit in the poorly invasive human oral SCC9 cell line increased cell migration on fibronectin and invasion through basement membrane by inducing MMP-3, and increased the cell growth in vitro and in vivo . Additioαvβ6 is expressed at the tips of migrating epithelial sheets during oral mucosal wound healing . It promTargeting αvβ6 integrin with an inhibitory peptide significantly inhibits the proliferation of oral squamous cell carcinoma cells . Xue et Expression of the β8 and β1 integrin subunits has been shown to be slightly higher in laryngeal SCC tissue than in normal epithelium , confirmMany studies have been carried out investigating the association of the β1 integrin subunit with HNSCC. The interpretation of these studies is complicated by the fact that β1 is widely expressed, and partners 12 α subunits to form members of all the integrin subfamilies: receptors for laminins, collagens, and RGD-containing ECM proteins. The β1 subunit has been proposed as a target in several of the studies described below. Development of small molecule drugs would be challenging because of the diverse range of receptors containing β1, and the potential normal tissue toxicity requires further investigation. β1 expression per se is more likely to be important as a biomarker rather than therapeutic target, which would require identification of the specific β1 integrins promoting pathology.β1 expression has been associated with HNSCC metastasis in multiple models. β1 integrin subunit levels were significantly higher in primary tumours which had metastasised than in non-metastatic tumours and its expression was inversely correlated with overall survival. Reduction in β1 integrin levels reduced lymph node and lung metastasis in vivo and matrigel invasion in vitro by preventing activation of MMP-2 . In oralβ1 , α6 99]99], and More details of the β1 integrin-interacting α integrin subunits in HNSCC became available recently. α11 is a biomarker of cancer associated fibroblasts and is overexpressed in HNSCC stroma . α2, α3,The remaining α subunit which interacts with β1 to form a RGD-binding integrin is α8. Little is known regarding its role in any cancer. One study has found α8 to be downregulated in laryngeal SCC compared to normal tissue .Expression of α5 in oesophageal SCC patient samples is significantly correlated with lymph node metastasis, tumour size and poor overall survival of oesophageal SCC patients, and expression increased in cancer cell lines compared to normal oesophageal epithelial lines . Oral SCKnockdown of α5 inhibits oesophageal SCC cells growth, migration, and invasion . A smallαIIbβ3 is the major integrin found on platelets and has been reported to be ectopically expressed in some human tumours and cancer cell lines ,111. It Targeting RGD-binding integrins for tumour imaging has been extensively studied. The majority of probes target αvβ3 , al18F-galacto-RGD as a PET imaging agent to detect tumours have included HNSCC [18F-galacto-RGD was proposed as an imaging agent for use in selection and monitoring of patients treated with antiangiogenic therapies. 111In-RGD2 has been investigated preclinically and showed promising results in selectively imaging tumour angiogenesis [Clinical trials of ed HNSCC ,120,121.ed HNSCC , and 18Fogenesis .TM 800, a labelled RGD tetramer, to facilitate removal of residual tumour tissue in an orthotopic HNSCC mouse model. NIR-guided surgery increased the recurrence free survival rate by 50%. This orthotopic model allows long term follow up of mice, but the CAL33 tumour cell line used was highly αvβ3-expressing which does not represent HNSCC clinical integrin expression profiles [Non-radioactive imaging modalities based on RGD are also in development. RGD-bound near-IR emitting quantum dots allowed non-invasive imaging of xenografts up to 12 hours after injection, with the best image intensity obtained up to 6 hours after treatment . Other nprofiles . The samprofiles . An indoprofiles .The overexpression of integrins on tumour cells or associated vasculature can be used to selectively deliver a cytotoxic agent to the tumour, thereby reducing toxicity to normal tissue. The general principles and breadth of integrin-targeted drug delivery have been reviewed extensively elsewhere ,126,127.Specific to targeting HNSCC, Tsien et al. ,131 have16)2-Glu-C2-KSSPHSRNSGSGSGSGSGRGDSP designed to contain both the RGD and PHSRN sequences from fibronectin required for high α5β1 affinity to deliver iron oxide particles containing a photodynamic therapy agent [αvβ3 is the most popular target for drug delivery, but it is not the only receptor targeted. Agents have been targeted to β1 using peptide  in normal tissue. The evidence to date clearly shows that integrin inhibitors can be considered molecular targeted anticancer therapy. αvβ3 appears most suitable for development of imaging agents to improve identification of margins for surgical resection of tumours. Overexpressed integrins will also be useful for targeted delivery of therapeutic agents. To date, αvβ3 and α5β1 have been targeted but the characteristic αvβ6 overexpression seen in HNSCC tumour cells  should aAlthough published data on αvβ5 is conflicting (see ,59,60 an