TREM2) is linked to dementia and neurodegenerative disease. It encodes expression of an innate immune receptor in the brain expressed by microglia. The level of the soluble fragment of TREM2 (sTREM2) is reported to increase in the cerebrospinal fluid (CSF) already in prodromal and asymptomatic Alzheimer’s disease.Delirium and dementia share symptoms of cognitive dysfunctions, and mechanisms of neuroinflammation appear involved in both conditions. Triggering receptor expressed on myeloid cells 2 (n = 120), and some of the patients developed delirium (n = 65). A medical delirium cohort (n = 26) was also examined. ELISA was used to determine the level of sTREM2 in CSF.We analyzed the level of CSF sTREM2 in relation to delirium and dementia. The study included patients with or without pre-existing dementia who underwent acute hip fracture surgery , particularly among patients developing delirium after CSF sampling . Between patients with dementia, there was no group difference, but the CSF sTREM2 level increased with waiting time for surgery  and correlated well with the CSF Alzheimer’s disease biomarkers, Aβ42, and t-tau/p-tau . Among patients with dementia, the level of Aβ38 and Aβ40 also correlated positively with sTREM2 in CSF .Delirium was associated with a higher level of CSF sTREM2 only among those without pre-existing dementia  contains supplementary material, which is available to authorized users. Delirium is an acute state of confusion with fluctuating symptoms of disturbed attention and cognition commonly precipitated by stress, such as surgery, in frail patients . BesidesWhile delirium increases the risk of dementia, dementia is also a delirium risk factor –8. The mTREM2 are known as dementia risk factors ).The initial hip fracture trauma presumably activated peripheral immune responses in the patients. The neuroinflammatory hypothesis of delirium suggests that brain dysfunctions and clinical presentation are secondary to peripheral immune activation . DementiExploring relations between CSF biomarkers may provide better pathogenic understanding by hinting to simultaneously ongoing processes in the brain. CSF Aβ and t-tau/p-tau markers related positively to CSF sTREM2 only in dementia patients with delirium, possibly suggesting somewhat distinct biological processes of delirium with or without pre-existing dementia. CSF sTREM2 related positively to CSF Aβ42 in the demented patients, essentially all of which were below the CSF Aβ42 cut-off level. Clinical data suggest that Aβ42 is sequestered by senile plaques, long before symptom onset leading to a pronounced drop in CSF Aβ42. This low CSF Aβ42 level is then stable in the individual patient , 48. ThuAn expanding literature links Aβ production to neuronal and synaptic activity and biological rhythms, e.g., sleep-wakefulness , 54. We CSF sTREM2 and tau markers interrelated in several studies of AD cohorts, yet in different subpopulations , 20, 58.With aging, dementia does not well relate to Aβ pathology and tauopathy, presumably because of increased importance of cerebrovascular comorbidity . The higThe present biomarker analyses hint at the biological processes of delirium being dependent on pre-existing dementia with selectively increased CSF sTREM2 in delirium only in the absence of dementia. These preliminary findings need replication in independent patient cohorts and assays. Statistical power is a study limitation with small cohorts after stratification. CSF sampling aged frail patients in a confused state is practically and ethically difficult and the CSF material used is unique in size and composition. As judged by mutant TREM2-variants, both increased and decreased shedding can reflect reduced cell surface TREM2 and impaired gene function , 60. IncAdditional file 1:Figure S1. CSF sTREM2 and the relation to age in hip fracture patients. Figure S2. Triplex MSD measurements of CSF Aβ peptides in dementia patients. Table S1. CSF t-tau/Aβ42 and p-tau/Aβ42 ratios and correlations to CSF sTREM2 in the hip fracture cohort. Figure S3. CSF sTREM2 and the ratios of t-tau/Aβ42 and p-tau/Aβ42. Figure S4. CSF sTREM2 and age in medical delirium patients (PDF 4106 kb)


Glioblastoma is a malignant brain tumor with mean overall survival of less than 15 months. Blood vessel leakage and peritumoral edema lead to increased intracranial pressure and augment neurological deficits which profoundly decrease the quality of life of glioblastoma patients. It is unknown how the dynamics of cerebrospinal fluid (CSF) turnover are affected during this process. By monitoring the transport of CSF tracers to the systemic blood circulation after infusion into the cisterna magna, we demonstrate that the outflow of CSF is dramatically reduced in glioma-bearing mice. Using a combination of magnetic resonance imaging (MRI) and near-infrared (NIR) imaging, we found that the circulation of CSF tracers was hindered after cisterna magna injection with reduced signals along the exiting cranial nerves and downstream lymph nodes, which represent the major CSF outflow route in mice. Due to blockage of the normal routes of CSF bulk flow within and from the cranial cavity, CSF tracers were redirected into the spinal space. In some mice, impaired CSF clearance from the cranium was compensated by a lymphatic outflow from the sacral spine. Glioblastoma is one of the most vascularized cancers. The newly formed blood vessels are leaky with a disrupted blood-brain-barrier (BBB), which leads to the formation of peritumoral edema and increased intracranial pressure3. Neurological symptoms and signs exacerbated by this edema dramatically reduce the life quality of glioblastoma patients4.Glioblastoma arises from astrocytes and is classified as World Health Organization Grade 4 infiltrative glioma. The mean overall survival achieved with repeat surgery or irradiation or with salvage chemotherapy remains only 14.6 months5. Lymphatic vessels have never been identified within the parenchyma of the central nervous system (CNS). Thus, outflow of CSF was historically assumed to directly enter the blood through arachnoid villi that project into the dural venous sinuses6. However, using a recently developed near-infrared (NIR) imaging method that allowed us to evaluate the dynamics of transport to systemic blood of tracers injected into the lateral ventricle or cisterna magna in mice, we discovered that return of CSF to the systemic blood is predominantly through the lymphatic vasculature in this species. Consistent with several earlier reports8, CSF tracers exited along perineural routes through foramina of the skull to reach lymphatic vessels outside the cranium9.Cerebrospinal fluid (CSF) is produced mainly by the choroid plexus lining the ventricles and partially from interstitial fluid (ISF) from the brain tissue produced at the BBB. CSF flows through the ventricular system to the subarachnoid space surrounding the brain and spinal cord12. However, it is not yet clear how CSF outflow is altered during pathological conditions, such as the vasogenic edema that occurs in glioblastoma. It is conceivable that similar to the acute conditions, CSF outflow would be accelerated in glioblastoma due to the intracranial hypertension and brain edema. However, edema may alternatively lead to a slower turnover of CSF due to reductions in CSF formation and/or blocked outflow routes. Therefore, it is highly interesting and clinically relevant to study the changes in CSF outflow that occur in glioblastoma. This might potentially lead to novel interventions for brain tumor induced edema, improve the drug delivery in glioblastoma patients and provide insights into glioblastoma antigen drainage.The outflow of CSF varies between physiological and pathological conditions. As expected from a bulk outflow system, the dynamics of CSF outflow are accelerated under conditions of acute rises in intracranial pressure such as the introduction of large CSF infusion volumesIn this study, we used the GL261 glioma mouse model and employed dynamic NIR imaging and magnetic resonance imaging (MRI) to monitor the CSF distribution and outflow after the infusion of tracers into the cisterna magna. Contrary to our expectations, we found that CSF outflow is significantly reduced in glioma-bearing mice. Dynamic imaging of tracer signals showed that the transport of tracers to the blood was delayed with overall signals at 60 min after tracer infusion significantly lower in glioma-bearing mice. Tracers signals at the perineural outflow routes from the skull were significantly lower in glioma-bearing mice compared to non-tumor bearing mice. By MRI, we found that the tracer in glioma-bearing mice was retained at the cisterna magna injection site compared to non-tumor bearing mice. Surprisingly, in some glioma-bearing mice, we detected high tracer signal in the spinal region. Mice showing high tracer signal in the sacral region of the spine demonstrated lymphatic outflow to iliac lymph nodes and more tracer transport to systemic blood, indicating that CSF may be redirected in glioma-bearing mice to spinal lymphatic outflow pathways after the blockage of cranial CSF outflow routes.3 were excluded from all further studies. We also confirmed brain edema14 as indicated by the wet-to-dry weight ratio of the whole brain 14 days after GL261 cell injection. As expected, the degree of brain edema was significantly higher in glioma-bearing brains  and sagittal  direction at 15 min, 25 min, 35 min, 45 min and 55 min after infusion Fig. . Using t15. To map out the tracer distribution within CSF, we perfused P40D680 NIR tracer into the cisterna magna and imaged ex vivo the basal aspect of the brain and the dorsal surface of the hemisphere contralateral to the glioma/PBS inoculation side of the brain. The tracer intensity in the basal aspect of the brain was 3 times weaker in the glioma mice compared to controls  and Prox1-GFP reporter mice2)47.GL261 cells were maintained in DMEM supplemented with l-glutamine, 10% FBS, and penicillin/streptomycin under standard culture conditions  30 mins before the procedure. The mice were anesthetized by inhalation of 2.5% isoflurane and fixed in a stereotaxic frame  on a heating pad (37 °C). The skull was thinned with a dental drill (RWD) at a location 2 mm lateral and 2.5 mm caudal to the bregma. A single cell suspension of 20,000 GL261 cells was prepared in 2 µl of PBS and injected at a speed of 1 μL/min with a 26 G needle into the right striatum at a depth of 3 mm from the skull surface. The needle was left in place for 2 minutes and then slowly removed while observing if any significant backflow occurred. The injection hole was closed with bone wax (ETHICON) and the scalp was sutured and glued with tissue glue (Histoacryl). The mice were observed in a cage with a heating pad at 37 °C until fully recovered from anesthesia.15. A skin incision was made over the occipital bone and the covering muscle layers were carefully dissected to expose the atlanto-occipital membrane covering the cisterna magna. A glass capillary with a diameter of 30 ~ 50 µm was used to penetrate the membrane perpendicular to the dura. For the MRI experiments, 5 µL Gadospin D solution  was infused at the speed of 1 µL/min. For the NIR imaging experiments, 2.5 µL of 200 µM P40D680 was infused at the speed of 0.5 µL/min. After the infusion, the capillary was left in place for 5 min and tissue glue (histoacryl) was applied at the injection site. The tip of the glass capillary was then cut off by spring scissor and the wound was closed by tissue glue.Mice were anesthetized by intraperitoneal injection of 80 mg/kg ketamine and 0.2 mg/kg medetomidine, fixed in a stereotaxic frame (RWD) and maintained at 37 °C body temperature using a heating pad (Stoelting). The injection was performed as described in a previous study21, fur above the saphenous vein region was removed with a razor and depilation cream before the tracer infusion. After closure of the wound after tracer infusion, the mice were immediately positioned under a Zeiss StereoLumar.V12 stereomicroscope with AxioVision software  and a Photometrics Evolve 512 camera  in a supine position on a heating pad to retain body temperature. The autofluorescence signal on the GFP channel was used to position the saphenous blood vessels at 25× zoom. Dynamic imaging was initiated 10 minutes after completion of the cisterna magna infusion by acquisition of a sequence of images (1 image every 15 s for 50 min) with a Cy5 filter set to monitor the NIR signal of the saphenous vein. Exposure time and camera gain settings were 200 ms and 200, respectively.For noninvasive imaging of tracer signals in the bloodTo assess the tracer transport to the systemic blood, a circular region of interest with a radius of 100 μm was placed over the saphenous vein on the acquired videos. Using the Measure Profile function, a table of fluorescence intensity in counts versus time was exported into Microsoft Excel. Since there was a loss of signal at the beginning of the scans due to photobleaching of tissue autofluorescence, baseline intensity in counts was calculated as the average signal of the lowest ten consecutive imaging frames. This baseline intensity was then subtracted from the fluorescence intensity values in order to plot fluorescent signal enhancement versus time in minutes. The NIR fluorescent signal enhancement value in counts at t = 60 min and the transit time in minutes of the arrival of tracer to the blood circulation  were assessed.in situ at 25× and 200 ms exposure time. Since there were no apparent differences in signal in the lymph nodes on the injected and contralateral sides, the average value of the nodes on each side was used for statistical analysis. The thoracic and sacral regions of the spine were imaged after removal of the skin at 15× and 200 ms exposure time. The region of the thoracic image starts from the junction point of the muscle spinalis et semispinalis thoracis and muscle longissimus thoracis and extends in a caudal direction with the spine in the middle of the image. The region of the sacral region image starts from the base of the tail and extends in a rostral direction with the spine in the middle. For analysis of signal enhancement in the analyzed tissues, background signals were subtracted based on the average signal of 3 uninjected mice.Images of P40D680 tracer spread on the basal and dorsal aspects of the brain, within the CNS-draining lymph nodes and within the spinal column were acquired with a Zeiss AxioZoom V16 microscope and a QImaging OptiMOS sCMOS camera  combined with a light-emitting diode illumination system pE-4000  and ZEN 2 software . In excised brains, images were acquired over the contralateral dorsal hemisphere  and on the entire ventral side of the brain . Images of lymph nodes were acquired 48 with an echo time/repetition time = 2.5/12.0 ms, flip angle 25°, matrix 480 × 360 × 180, field of view 36 mm × 27 mm × 18 mm, zero fill 2, 1 average and a scan time of 3 min 46 s. Both horizontal  and sagittal  sections were scanned at t = 15 min, 25 min, 35 min, 45 min, 55 min after Gadospin D infusion. Signal intensity (SI) reduction in blood vessels was achieved by placement of a saturation slice over the mouse heart. SI and noise were measured with ImageJ (Version 2.0.0). The ROI placements and the signal over noise ratio calculation were shown in Fig. To measure the contrast agent distribution after cisterna magna infusion, mice were anesthetized and 5 µL Gadospin D  infused at a speed of 1 µL/min. Immediately after injection, the head of the mouse was fixed in the stereotactic frame attached to a 9.4 T animal scanner (Bruker Biospin GmbH) with a BGA12S gradient system with ParaVision 6.0.1 (Bruker Biospin GmbH). A four–element receive–only cryogenic phased array coil (Bruker BioSpin GmbH) was used in combination with a linearly polarized room temperature volume resonator for transmission. Imaging was performed with a three-dimensional time of flight gradient recalled echo sequence (3D-TOF-GRE)To determine the glioma volume, glioma-bearing mice were anesthetized by inhalation of 2.5% isoflurane and injected with the contrast agent Dotarem  subcutaneously 5 mins before imaging. The measurement was performed in a 7 T animal magnetic resonance imaging (MRI) system (Bruker BioSpin GmbH). A four–element receive–only cryogenic phased array coil (Bruker BioSpin GmbH) was used in combination with a linearly polarized room temperature volume resonator for transmission. The mice were stereotactically fixed and the body temperature maintained at 37 °C. A rapid acquisition with relaxation enhancement (2D TurboRARE) sequence was used with the following parameters: echo time/ repetition time = 36/2200 ms, rare factor = 8, BW = 50 kHz, FOV 15 mm × 15 mm, matrix 256 × 256, slice thickness = 0.5 mm, 13 slices, 3 averages, scan time = 2 min 38 s. The scans were analyzed with ImageJ (Version 2.0.0). The volume of the glioma was calculated as the sum of glioma area in each slide multiplied by the slide thickness.Statistical analyses were performed with GraphPad Prism5 (GraphPad Software Inc.). Graphs represent mean ± SD. Student’s t-test was used to compare two groups. Other types of tests are indicated in the figure legends. A p-value < 0.05 was considered statistically significant (indicated by asterisks).All mouse experiments were approved by the Kantonales Veterinaramt Zurich (license numbers 185/13 and 161/16) and performed following the regulations of the Swiss Federal Welfare Act (TSchG).Supplementary Information


Escherichia coli donor and a Salmonella Typhimurium recipient were co-incubated for 4 h in media containing different levels of the stress factors’ pH, osmolality, copper, zinc and acetic, propionic, lactic, and n-butyric acid, as well as subtherapeutic levels of cefotaxime, sulfamethoxazole/trimethoprim, and nitrofurantoin. Conjugation frequencies were calculated as transconjugants per donor, recipient, and total bacterial count. A correction factor for the stress impact on bacterial growth was used. Acetic, lactic, and n-butyric, acid, as well as pH, showed no significant impact. In contrast, increasing concentrations of propionate, zinc, copper, and nitrofurantoin, as well as increased osmolality reduced conjugation frequencies. Sulfamethoxazole/trimethoprim and cefotaxime showed increased transconjugants per donor, which decreased after correction for stress. This study showed, for the model mating pair, that conjugation frequencies decreased under different physiological stress conditions, and, thus, the hypothesis that stress factors may enhance conjugation should be viewed with caution. Furthermore, for studies on in vitro gene transfer, it is vital to consider the impact of studied stressors on bacterial growth.The transfer of extended spectrum β-lactamase (ESBL)-genes occurs frequently between different bacteria species. The aim of this study was to investigate the impact of nutrition related stress factors on this transfer. Thus, an  Enterobacteriaceae (ESBL-PE) pose a major hazard on public health, the development of methods to reduce their occurrence has gained high priority [Escherichia coli being the most common species [E. coli isolates may also inhabit the intestinal tract of broilers as part of the commensal microbiota [E. coli (ESBL-EC). This indicates that ESBL-EC may be harmless and does not require antibiotic treatment. However, as ESBL-genes are frequently located on plasmids, which are commonly transmitted between Enterobacteriaceae [Enterobacteriaceae. This can induce the spread of antibiotic resistant pathogens in livestock.As extended-spectrum β-lactamase (ESBL)-producing priority ,2. In li species . Besidescrobiota ,5, and Eeriaceae , these pThus, animals may develop a disease, which is difficult to treat by antibiotics. Furthermore, treatment with β-lactam antibiotics may additionally increase the ESBL-PE load by selective pressure ,7,8. ESBT stands for the number of transconjugants/mL, R for the number of recipients/mL, D for the number of donors/mL, and T, D, and R will range between 102 and 109, in most experiments the time will stay below 100. Thus, mathematically, time differences will not be large enough to affect the result. Additionally, these results are not easily compared with the results calculated with the more traditional approach of dividing transconjugants by donor or recipient counts. Furthermore, a prerequisite for this equation is that donor and recipient concentrations remain rather constant during the period of time. This assumption does not apply for the intestinal microbiota. In the intestinal tract, bacteria undergo dynamic growth states with a constant change of the composition [Bacteria, which transfer mobile genetic elements to other bacteria, are referred to as donors, while bacteria capable of acquiring the genetic information are termed recipients. Once the plasmid is successfully transferred, the recipient, harboring the ‛new’ plasmid, will be termed transconjugant . As the nors/mL,,16,17., harboring the CTX-M-1bla gene and belonging to the B1 subgroup, was chosen as the donor. The strain Salmonella Typhimurium L1219-R32 (isolated from pigs) was chosen as the recipient strain. This mating pair was revealed to be the best fit for the study design in a previous study obtaining conjugation kinetics for five potential E. coli donor strains and six potential Enterobacteriaceae recipients every 2 h for 22 h [5 transconjugants/donor after 4 h of co-incubation  [E. coli incubation or 300 µg/mL sulfamethoxazole/trimethoprim (SXT)  for the Salmonella Typhimurium strain. All strains were incubated aerobically at 37 °C.A nonpathogenic for 22 h . From thditions) . All cul8 cells/mL. Fifty µL of each donor and recipient strain were added to 800 µL media supplemented with stress factors, as described below. After vigorous vortexing, the samples were incubated aerobically for four hours at 37 °C. The suspensions were then placed on ice, serially diluted, and spread on a MacConkey agar  containing 8 µg cefotaxime/mL and 300 µg sulfamethoxazole/trimethoprim/mL to obtain transconjugants and on MacConkey agar without antibiotics to estimate the cell count of E. coli ESBL10682, Salmonella Typhimurium L1219-R32, and total bacterial count (TBC). In the set up with cefotaxime, MacConkey agar plates containing 8 µg cefotaxime/mL, or 300 µg sulfamethoxazole/trimethoprim/mL were used to obtain the cell count of E. coli ESBL10682 and Salmonella Typhimurium L1219-R32, respectively. The conjugation frequency was calculated with respect to the donor, the recipient, and the total bacterial count by dividing the number of transconjugants/mL by the respective bacteria count:After the second preculture in antibiotic supplemented medium, the bacterial strains were washed twice in Phosphate Buffered Saline (PBS)  and diluted to 5 × 10All experiments were repeated three times with fresh cultures and with three replicates per repetition.d/l-lactate. For the challenge experiments, various stress factors were added to Mueller Hinton 2 broth in different concentrations. The studied stress factors were pH, osmolality, antibiotics at subtherapeutic concentrations, zinc, copper, and the short chain fatty acids acetic, propionic, and n-butyric acid and The impact of pH 4–7.5 on donor and recipient growth was determined by measuring turbidity during incubation in a micro titer plate reader  at 690 nm every 5 min over a time period of 4 h (data not shown). A pH adjustment to 5.0, 5.5, 6.0, and 6.5 was carried out in a double concentrated Mueller Hinton 2 broth using 1 M hydrochloric acid (HCl) . Equal volumes were achieved by adding ultrapure water to the solutions in volumetric flasks. The media were then sterile-filtered . Mueller Hinton 2 broth exhibited a pH value of 7.5 and served as the control. Sodium chloride (NaCl)  was added to a 50 mL Mueller Hinton 2 Medium in Afnor bottles to obtain osmolalities of 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mOsm/kg and autoclaved. The correct osmolality was confirmed with a micro osmometer , and the impact on bacterial growth was monitored by a turbidity measurement at 690 nm. Correlation between osmolality and CF was analyzed using the software IBM SPSS . The osmolality of 300 mOsm/kg served as the control, as it resembled the osmolality of the pure medium.Subtherapeutic levels of nitrofurantoin (F) , cefotaxime, and sulfamethoxazole/trimethoprim were determined for the donor and recipient strains by studying their growth kinetics in the presence of different antibiotic concentrations. The impact on bacterial growth was monitored by turbidity measurement at 690 nm, measured for 4 h as described above. The antibiotics were added to Mueller Hinton 2 broth at each three different concentrations  while the Mueller Hinton 2 broth without antibiotics served as control. 4(H2O)5  were prepared according to Liedtke and Vahjen [Saturated solutions of zinc from ZnO  and copper from CuSOd Vahjen . Atomic d/l-lactic acid  and n-butyric acid  were added to double concentrated Mueller Hinton 2 Broth. The pH was adjusted to pH 7.5 using 5 M sodium hydroxide (NaOH) . The dilutions were sterile-filtered (0.2 µm) and the concentrations were confirmed by gas chromatography . Four different concentrations were prepared by 1:2 serial dilutions in Mueller Hinton 2 Broth  and the exact concentrations obtained by gas chromatography. The impact on donor and recipient growth was studied prior to the conjugation experiment ). The control medium was nonsupplemented Mueller Hinton 2 Broth.Acetic acid , propionic acid , ctr) and a certain level of supplementation with a stress factor (cfustress) was determined asSince stress does not only influence the conjugation but also the growth of the transconjugants, donors, and recipients, the results were corrected by a stress impact factor. This factor was defined as the percentage change of growth between the two concentrations/levels. At first, the stress impact factor (SIF), defined as the ratio between the mean colony forming units per mL of the control (cfucorr) to the level of the controls. Thus, a condition without an impact of the stressor on bacterial growth was simulated by multiplying the cfustress with the SIF: Thus, SIF = 1 would indicate no impact on the growth, SIF > 1 shows a reduction in growth, and SIF < 1 designates an enhanced growth when exposed to the stressor. Secondly, the growth of donors, recipients, and transconjugants were corrected . Results are presented as mean values ± standard deviation. The nonparametric Kruskal–Wallis test and Mann–Whitney test were used to determine significant differences and subgroups, respectively. Differences were considered statistically significant at A preliminary screening of the donor and recipient growth kinetics at various pH values failed to detect a significant impact on the donor or recipient strains at pH levels of 5.0–7.5. The lowest numeric conjugation frequency was noted at an initial pH of 6.0 for all alternative calculations, but differences were only marginal . BacteriSalmonella Typhimurium declined with increasing osmolality, while the E. coli strain showed the highest number of colony forming units at 500 mOsm/L  . Significant differences in the CF of 0.4–0.5 log cfu/mL were observed for all three approaches when CF was corrected by SIF (The growth of 0 mOsm/L . Conjugactively) . After cd by SIF .p = 0.06), with increasing concentrations of CTX when calculated based on donor count. This effect was profound for CF(R) and CF(T)  . When coIncreasing concentrations of sulfamethoxazole/trimethoprim decreased the growth of the donor strain by 1.8 log, while the growth of the recipient strain only increased with 0.2 log from no to 5 µg SXT/mL supplementation . FurtherDonor and recipient growth declined in a dose-dependent fashion at 0.7 and 0.8 log cfu/mL, respectively, with increasing concentrations of nitrofurantoin . SimilarE. coli ESBL10682 was not affected significantly by zinc supplementation, while Salmonella Typhimurium L1219-R32 declined by 0.6 log cfu/mL at the highest investigated zinc concentration compared to the controls  is frequently calculated by dividing the number of transconjugants/mL by the donor count per mL ,43,44,45The impact of different agents on conjugation events is commonly displayed as a change in CF. However, these agents do not only impact the formation of transconjugants, but also their growth and viability, as well as the growth and viability of recipient and donor. This creates a bias neglected by the common methods to calculate the CF as transconjugants/donor or transconjugants/recipient . If the From the above it is concluded that the method of calculation has a significant impact on the results, and, thus, different methods should be considered in studies on the impact of stressors on conjugation frequency. E. coli donor and a Salmonella Typhimurium recipient where an Inc GpI1 plasmid was transferred at a pH value of 4.3 [E. coli donors and recipients [In the gastrointestinal tract (GIT) of poultry, pH levels range mainly between 5 and 8, if gizzard and proventriculus are neglected ,47. The e of 4.3 . Similarcipients . Unfortucipients . This sucipients . HoweverOsmolality in the gastrointestinal tract of broilers varies between individual bowel segments. Accordingly, osmolalities of 540 (crop), 312 (gizzard), 571 (duodenum), 650–573 (jejunum), and 514–451 (ileum) mOsm/kg were reported , descendEnterobacter donor to an E. coli recipient under detection limit [Enterobacteriaceae in the presence of different metal surfaces with an E. coli and a Klebsiella pneumoniae donor and a E. coli recipient [4 and copper nanoparticles reduced conjugation frequencies [Since the ban of antibiotic growth promoters in the European Union (EC (No) 1831/2003), alternatives have especially gained importance . Zinc anon limit . As resuecipient . While Cquencies . From thquencies ,55,56,57quencies ,59. The Streptococcus thermophilus, Lactobacillus fermentum, Lactobacillus plantarum, and actobacillus bulgarLicus [Salmonella enterica serovar Typhimurium donor and recipient strains derived from mice in the presence of propionate [Enterobacteriaceae. Bacterial metabolites may also have an impact on bacterial physiology in vivo. For instance, a reduction of conjugation frequency was previously reported in the presence of lactic acid producing bacteria, such as lgaricus ,61,62,63opionate . As pH aLower numbers of donor cells would lead to a higher ratio of transconjugants per donor cell count.Lower numbers of recipient cells would lead to lower numbers of transconjugants and a decreased ratio of transconjugants per donor cell count .As recipient and donor are affected equally, CF will not differ significantly from control. The determined conjugation frequency depends on two factors—The bacterial concentration and the transconjugants’ growth. The bacterial concentration shapes the chance for donor and recipients to meet close enough to perform a plasmid transfer. On the other hand, transconjugant growth directly affects the number of detected transconjugants and thus the calculation and result of CF. Hence, it would be tempting to conclude that changes in CF can be explained solely mathematically due to variations of donor, recipient, and/or transconjugant concentrations in the presence of stressors. To investigate this further, an experiment with sublethal amounts of antibiotics was designed, to reduce the growth of (a) donor, (b) recipient, or (c) both donor and recipient. Assuming that conjugation comes at no fitness cost or gain, the transconjugants should grow similar to the recipients, as they are basically identical apart from their additional plasmid harboring resistance against CTX. If the explanation for the changes in CF were solely mathematical, the following situation would arise for the mentioned scenarios:This would lead to a lower number of transconjugants per donor in the osmolality and CTX experiments. This effect should be more profound for osmolality, since both the recipient count and transconjugant growth are affected negatively. CTX, on the other hand, reduces the growth of the recipient more than transconjugant growth. Thus, one expects decreasing CF(D), CF(R), and CF(T) with increasing concentrations of CTX and higher osmolality. Similarly, SXT supplementation would result in an increase of transconjugants per donor and higher CF(D), while lower CF(R) would be expected at increasing antibiotic concentration. Nitrofurantoin inhibited the growth of donors and recipients in an equal manner. Therefore, no significant differences were predicted. The results from the experiments differed from these assumptions. Thus, the changes in conjugation frequencies cannot solely be explained by changes in bacterial growth, justifying the conclusion that some stress factors may directly influence conjugation. E. coli donor to E. coli and Pseudomonas aeruginosa recipients had similar prerequisites as the CTX experiment, since donor growth was not affected as much as the recipient, which declined with increasing concentrations [Pseudomonas aeruginosa accepted the ESBL-carrying plasmids at a higher rate only at a state where the antibiotic concentration changed from low impact to high impact on recipient growth. The results agree with the results from the CTX experiment (CF(D)). In the case of the E. coli recipient, CF(D) decreased with increasing gentamycin [Antibiotics are frequently used in livestock as therapeutics, meta- and prophylaxis and, outside the European Union, in subtherapeutic levels to enhance performance ,10,65. Ttrations . Similartrations . Pseudomntamycin  supplementamycin . The impntamycin . Even afntamycin . CF(D) iIn conclusion, a negative impact on conjugation frequency was observed for osmolality, zinc, copper, and propionic acid, as well as subtherapeutic levels of antibiotics. No effects were found for pH or the bacterial metabolites lactate, acetate, or n-butyrate. Furthermore, no stressors increased conjugation frequency, and, thus, the hypothesis that stress generally increases bacterial conjugation should be viewed with caution. The results also show that, in studies focusing on stress related effects on gene transfer, the calculation of conjugation frequency should include the impact of a stressor on donor, recipient, and transconjugant. Still, it must be considered that the observed impact on conjugation frequencies might be strain specific. Future studies should, therefore, investigate if these observations can be repeated with different donor and recipient strains. In the present study, the impact on conjugation events was investigated for one stressor at a time. However, the intestinal tract of broilers combines these and further stressors. Thus, further studies should be anticipated to examine conjugation events in complex systems.


Pancreatic cancer is the fourth leading cause of cancer death in the United States and is increasing in incidence with a dismal prognosis . Treatmevs. 180 days for controls [in vitro and in vivo [via ascorbate radical formation, with ascorbate as the electron donor [Ascorbate (vitamin C) is one of the early unorthodox therapies for cancer, based on unsupported hypotheses promoted by Cameron and Pauling. A prospective study was conducted that randomized patients to ascorbate treatment or palliative therapy with treated patients having a median survival of 343 days controls . To testcontrols , 7. Subs in vivo . Pharmacon donor . More reon donor . In addion donor .What do we have to lose? Pharmacological ascorbate is safe with little side effects and can be given as an intravenous infusion as other chemotherapeutic treatments. A selective radiosensitizing (pro-oxidant) drug with a protecting effect (antioxidant) on surrounding tissue is novel and could potentially change current radiation dose limitations used, allowing more aggressive treatment of locally advanced pancreatic cancer. Clearly, phase II trials are needed and should be supported.


Pythium insidiosum is a member of the oomycetes class of aquatic fungus-like microorganisms. It can infect humans and animals through skin wounds and the eyes, causing pythiosis, an infectious disease with high morbidity and mortality rates. Antifungal agents are ineffective as pythiosis treatments because ergosterol, the target site of most antifungal agents, is not found in the P. insidiosum cytoplasmic membrane. The best choice for treatment is surgical removal of the infected organ. While natural plant products or secretory substances from bacterial flora have exhibited in vitro anti-P. insidiosum activity, their mechanism of action remains unknown. Therefore, this study hypothesized that the mechanism of action could be related to changes in P. insidiosum biochemical composition  following exposure to the inhibitory substances. The biochemical composition of P. insidiosum was investigated by Synchrotron radiation-based Fourier-transform infrared (FTIR) microspectroscopy.P. stutzeri ST1302, fraction No.1 from the crude extract of K. pneumoniae ST2501 and xanthyletin were used as anti-P. insidiosum substances, with MFCs at 3.125, 1.57–1.91, 0.003 mg/ml, respectively. The synchrotron FTIR results show that the deconvoluted peak distributions in the amide I, amide II, and mixed regions were significantly different between the treatment and control groups.Fraction No.6 from the crude extract of P. stutzeri ST1302 and K. pneumoniae ST2501 exerted anti-P. insidiosum activity that clearly changed the proteins in P. insidiosum. Further study, including proteomics analysis and in vivo susceptibility testing, should be undertaken to develop a better understanding of the mechanism of anti-P. insidiosum activity.Xanthyletin and the secondary metabolites from  Pythium insidiosum is a member of the oomycete class of aquatic fungus-like microorganisms. It can infect both humans and animals through the eyes or skin wounds to cause pythiosis, a disease with severe morbidity and high mortality rates [P. insidiosum lacks ergosterol in the cytoplasmic membrane, the target site of antifungal agents, which act by interfering with ergosterol synthesis and its function in the membrane [P. insidiosum activity. A new furanocoumarin isolated from the fruit of Scaevola taccada and secondary metabolites from Pseudomonas stutzeri and Klebsiella pneumoniae that were collected from swampy areas have also been found to exhibit in vitro inhibitory effects on the human pathogenic oomycete P. insidiosum [ty rates , 2. Hostty rates , 3, 4. Tty rates , 5. Mostmembrane . The mosmembrane . Interessidiosum , 8. NoneP. insidiosum after the organism was exposed to inhibitory substances, based on the presumption that these changes would reflect the mechanism of its inhibitory activity.Synchrotron Fourier-transform infrared (FTIR) microspectroscopy utilizes infrared (IR) radiation from a synchrotron light source that is transmitted through an interferometer to irradiate a sample and cause biomolecular vibrations. Biomolecules have unique vibrational characteristics which correspond to specific infrared light frequencies that are related to their functional groups –11. FTIRP. stutzeri ST1302 and K. pneumoniae ST2501 were divided into 14 and 5 fractions, respectively. Fraction No.6 (yellow solid) of the crude extract from P. stutzeri ST1302 was eluted with 20% methanol in dichloromethane, while fraction No.1 (brown semisolid) of the crude extract from K. pneumoniae ST2501 was eluted with 100% dichloromethane gave the strongest anti-P. insidiosum activity. Both the crude extract and fraction No.1 from the crude extract of K. pneumoniae ST2501 gave stronger anti-P. insidiosum activity and stability results than P. stutzeri ST1302.The crude extracts from P. stutzeri ST1302 and K. pneumoniae ST2501 showed anti-P. insidiosum activity against all eleven of the clinical strains of P. insidiosum by disc diffusion method. The MFCs were 3.125, 1.57–1.91 and 0.003 mg/ml for fraction No.6 from the crude extract of P. stutzeri ST1302, fraction No.1 from the crude extract of K. pneumoniae ST2501, and xanthyletin, respectively  of the 5 groups . The average second derivative and EMSC spectra showed an amide I peak at 1652 cm− 1, 1537 cm− 1 of amide II and P=O stretching vibration of phospholipids at 1243 cm− 1. Moreover, the spectral windows from 1800 to 1200 cm− 1 exhibited seven peaks, including at 1714, 1652, 1591, 1537, 1483, 1297 and 1243 cm− 1 which are different points in the five spectra groups.Fig. − 1 as a fatty acid region corresponding to lipid membrane, 1800–1500 cm− 1 as an amide I and II region corresponding to proteins and peptides, 1500–1200 cm− 1 as a mixed region containing vibrations of fatty acids, proteins, and polysaccharide, and 1200–900 cm− 1 as a polysaccharide region corresponding to carbohydrates. Each sample showed absorption bands in all four areas. We found significant differences between the treated and the control groups, in both the amide I and amide II region . The two dimensional of PC scores plot (PC1 and PC2) showed 68% of the total variance. The PC1 (55%) was reliable for the separation of P. insidiosum from the treatment groups, the positive control and the negative control. The PC2 (13%) explains the variation within each group. The loadings plot revealed a significant peak shift at the amide I (1646 cm− 1 shift to 1621 cm− 1) and amide II (1546 cm− 1 shift to 1523 cm− 1), representing the effect of thimerosal and anti-P. insidiosum on the amide I and amide II structure of P. insidiosum.Fig. − 1). The two dimensional of PC scores plot (PC1 and PC2) showed 61% of the total variance. The PC1 (44%) was responsible for the separation of P. insidiosum from the treatment and control groups, in which the positive and negative controls were distributed in the same area and were separated from treatment groups. The PC2 (17%) explains the variation within each group. The loading plot revealed a significant peak shift at the CH2 bending mode of lipids and proteins (1482 cm− 1 shift to 1459 cm− 1) and P=O asymmetric stretching vibrations in the head group of phospholipids (1239 cm− 1 shift to 1218 cm− 1).Fig. − 1). The two dimensional of PC scores plot (PC1 and PC3) showed 66% of the total variance. The PC3 was reliable for the separation of P. insidiosum from the treatment and control groups, in which the positive and negative controls were distributed in the same area and were slightly separated from the treatment groups. However, PC3 explained 10% of the separation of the sample groups, while PC1 showed 56% of the variation within each group. The PCA from the second derivative and EMSC spectra in the polysaccharides region (1200–900 cm− 1) couldn’t separate in any of the sample groups [Fig. e groups –15.Fig. P. insidiosum substances and the positive control, whereas the mixed regions containing lipids, proteins and phospholipids were changed in the three groups which were treated with xanthyletin and secondary metabolites from bacteria. No significant differences were found in the fatty acid and polysaccharide regions in all the sample groups.The PCA results of the four regions show that the protein components were changed in all the groups that were treated with anti-Pseudomonas stutzeri has been used to control fungal diseases in plants incited by Fusalium solani, Fusarium oxysporum, Fusarium moniliformae, Fusarium udum, Macrophomena phaseolina, Rhizoctonia solani, Colletotrichum capsicii, Aspergillus flavus, and Aspergillus niger [Klebsiella pneumoniae exhibits anti-fungal activity through secreted bioactive compounds that suppress the growth of Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Penicillium expansum, Aspergillus niger and Aspergillus terreus. Abeer et al. reported that 100 mg/mL of crude extract from K. pneumoniae gave higher diameter zones of inhibition than 30 μg/mL of amphotericin B, fluconazol and miconazole nitrate when used against Aspergillus flavus [K. pneumoniae ST2501 gave stronger anti-P. insidiosum activity and stability than the crude extracts and fractions from P. stutzeri ST1302. This was due to K. pneumoniae ST2501 producing many more important secondary metabolites with high biological activities when compared to P. stutzeri ST1302. Abeer Fauzi Al-Rubaye et al. [K. pneumoniae produces many important secondary metabolites with high biological activities as anti-fungal activity.Antagonistic microorganisms play a major role in microbial equilibrium and serve as powerful agents for biological disease control . Pseudomus niger . Moreoves flavus . In the e et al. , reportepyranocoumarins (coumarin derivatives) and has been shown to have anti-fungal activity against Candida albicans and filamentous fungi [. Of the tested compounds in the present study, xanthyletin had the strongest anti-P. insidiosum activity. The observed MFC of 0.003 mg/ml correlated well with compound 5 from the fruits of Scaevola taccada that showed anti-P. insidiosum activity with the MIC of 0.005 mg/ml [P. insidiosum hyphae is promising for the development of antimicrobial drugs against pythiosis.Coumarins are members of a class of compounds called benzopyrones, which consist of fused benzene and α-pyrone rings. Most coumarins are secondary metabolites of green plants, but some coumarins are produced by fungi and bacteria. Xanthyletin is a member of us fungi . Of the 05 mg/ml . A higheP. insidiosum hyphae by synchrotron radiation-based FTIR microspectroscopy that shows the spectral data specific to functional groups of biological molecules such as proteins, lipids, and carbohydrates. Statistical analysis of the FTIR spectra found protein changes in P. insidiosum when treated using thimerosal (positive control), the three sample groups treated with xanthyletin, and two anti-P. insidiosum substances from P. stutzeri ST1302 and K. pneumoniae ST2501. However, the pattern of the proteins changes in the positive control and the three treated sample groups were different. Thimerosal’s anti-microbial action is dependent on it breaking down and releasing ethyl-mercury to penetrate the cell membranes and binds to intracellular enzymes, which then inhibits them and causes cell death [P. insidiosum, but the MICs are incompatible with safe plasma levels [P. ultimum: reduced incorporation of amino acids into proteins, inhibition of protein synthesis, and inhibition of amino acid transport [P. stutzeri ST1302 and K. pneumoniae ST2501 might be protein components of this organism.Synchrotron radiation-based FTIR microspectroscopy was used to for the identification of filamentous fungi along with analyzed spectra data and statistical analysis of biomolecular discrimination , 10. Thill death . Interesa levels , 22. Whiransport . TherefoP. stutzeri ST1302 and K. pneumoniae ST2501 showed anti-P. insidiosum activity, characterized by clearly changed proteins in P. insidiosum. Future study should seek to include proteomics analysis to improve understanding about the mechanism of anti-P. insidiosum activity and an in vivo study of these anti-P. insidiosum extracts.In vitro studies of xanthyletin and the secondary metabolites from Pythium insidiosum preparation: Eleven clinical strains of P. insidiosum, isolated from the ocular, vascular, and disseminated forms of human pythiosis were identified by Assoc. Prof. Dr. Angkana Chaiprasert, Department of Microbiology, Mahidol University. These organisms were rechecked by culture on Sabouraud Dextrose Agar at 25 °C, detect perpendicular sparsely septate hyphae under the light microscope, induced zoospore formation and PCR identification [fication . The culPseudomonas stutzeri ST1302 and Klebsiella pneumoniae ST2501 preparation. P. stutzeri ST1302 and K. pneumoniae ST2501 that produced anti-P. insidiosum substances were isolated from water sampling area around Khon Kaen University and identified by Dr. Yordhathai Thongsri [Thongsri . The mic14H12O3) powder as a coumarin compound was obtained from ChemFaces . It showed 98% of purity tested and 228.24 of molecular weight.Xanthyletin: Commercially xanthyletin (CP. stutzeri ST1302 and K. pneumoniae ST2501 inoculants were prepared to McFarland Standard No.1 (3 × 108 CFU/ml), 700 μl of this concentration was inoculated by aseptic technique into six 1000 ml bottles, each of which contained 700 ml of brain heart infusion (BHI) broth. Each bottle was then incubated on a rotary shaker at 200 rpm at 37 °C for 3 days. Then, 4.2 l of culture broth was centrifuged at 7500 rpm for 20 min at 4 °C and sterilized by filtration through a 0.45 μm pore size membrane filter (Millipore). Cell free filtrate was concentrated ten-fold in a rotary evaporator . Four hundred and twenty milliliter of the cultured broth was then mixed three times with a two-fold volume of dichloromethane (Fisher) for P. stutzeri ST1302, and ethyl acetate (Fisher) for K. pneumoniae ST2501. Each dichloromethane and ethyl acetate layer was then dried with anhydrous Na2SO4 (Merck) and concentrated in a rotary evaporator .The P. stutzeri ST1302 and K. pneumoniae ST2501 were fractionated by activity-guided separation liquid column chromatography (LCC). The extracts were applied to a glass column filled with a slurry of silica gel 60 , which had been preconditioned with dichloromethane for the crude extract from P. stutzeri ST1302, and with ethyl acetate for the crude extract from K. pneumoniae ST2501. Gradient mixtures of dichloromethane and methanol were used as mobile phases. The eluent was collected and fractions defined according to their thin layer chromatography (TLC) characteristics. TLC was using silica gel GF254 pre-coated aluminum sheets. The fractions containing the same major compounds were combined and tested for anti-P. insidiosum activity using the disc diffusion method described in the following section. The fraction with inhibitory activity from P. stutzeri ST1302 was separated again using LCC with gradient mixtures of dichloromethane and hexane (Merck) as mobile phases. TLC was then used for the differentiation of single fractions according to retention factors (Rf) and to check purity. Detection was under exposure to UV light at 256 and 364 nm.The crude extracts from P. insidiosum activity. The testing solutions were prepared as 500 mg/ml stock solutions. Paper discs  were placed onto SDA plates with P. insidiosum and grown for 2 days, and 20 μl of each testing solution was added to the discs (dichloromethane and ethyl acetate were used as a negative controls). The testing plates were stored at room temperature for 2 h in a laminar flow biosafety cabinet to testing the solution diffusion and they were then incubated at 37 °C for 3, 6 and 9 days. Inhibition zones were measured when the growth of P. insidiosum reached the negative control discs.Testing solutions, including the whole crude extract, fractions of the crude extract and xanthyletin, were tested for anti-P. insidiosum effect of the crude extract from the bacteria and xanthyletin, we used the method of Rodrigo Trolezi et al. with some modifications [P. insidiosum mycelia (5 mm in diameter) from the SDA plates were inoculated by aseptic technique into 1.95 ml of Sabouraud Dextrose Broth (SDB) and incubated at 37 °C for 3 days. Fifty microliters of varying concentrations of the testing solutions that had anti-P. insidiosum activity were added to the cultures and incubated at 37 °C for 24 h. Fifty microliters of organic solvent, which dissolved the anti-P. insidiosum substances, was added and incubated at 37 °C for 24 h as the negative control. The P. insidiosum fragments were then placed on SDA plates and incubated at 37 °C for 3, 6 and 9 days to follow the hyphal growth and determine the minimal fungicidal concentration (MFC). All tests were performed in triplicate.To investigate the in vitro anti-ications . BrieflyP. insidiosum treated by 0.02% (w/v) thimerosal was selected as the positive control, while P. insidiosum mycelia fragments in SDB plus organic solvent was selected as the negative control. After incubation, 1 ml of individual mycelia were collected and transferred to an Eppendorf tube and centrifuged (430 x g for 30 s) to remove the culture supernatant. The mycelia were then washed 3 times by 1 ml of 0.9% NaCl by centrifugation (430 x g for 30 s) and the supernatant was discarded. Mycelial pellets were resuspended in 300 ml of 0.9% NaCl before FTIR microspectroscopy analysis.According to the broth dilution susceptibility testing, the MFC of each testing solution was selected for FTIR microspectroscopy analysis. 2) window for synchrotron radiation-based Fourier transform infrared (FTIR) microspectroscopy investigation. The CaF2 windows were dried in a class II biological safety cabinet (BSC) for 30 min and kept in the desiccator until FTIR investigations to avoid the influence of water absorption on the infrared spectra. FTIR spectra were acquired using a Hyperion 2000 IR microscope coupling with a Vertex 70 spectrometer . The internal light source was replaced by synchrotron light delivered from the front-end of Beamline 4.1: IR at Synchrotron Light Research Institute (Thailand). The spectra were recorded in transmission mode using a 15x objective, 64 accumulations per sample, a spectral resolution of 4 cm− 1, and a spectral range of 4000–900 cm− 1. Before the samples were measured, the background spectrum of the CaF2 window was recorded and then subtracted from the sample signal. Pre-processing of infrared spectra included the region 4000–900 cm− 1, all spectral data were extracted by OPUS software .Five microliters of mycelia suspension were placed on a calcium fluoride (CaF− 1 as the fatty acid region (corresponding to lipid membrane), 1800–1500 cm− 1 as the amide I and II region (corresponding to proteins and peptides), 1500–1200 cm− 1 as the mixed region containing vibrations of free fatty acids, proteins, and phospholipids, and 1200–900 cm− 1 as the polysaccharide region (corresponding to carbohydrates). The second derivative was performed with Savitzky Golay 19-point smoothing which removed the broad baseline offset. In addition, the final spectra were taken as an extended multiplicative scatter correction (EMSC), which eliminated the physical information collected from the light scattering and removed artefacts that were introduced from normalizing the path length differences. This ensured that the spectra obtained would only be from the functional groups of the biomolecules. The EMSC corrected data is a robust representation of the biomolecules present. After the EMSC processing, the samples were classified using principal component analysis (PCA).Principal Component Analysis (PCA) was performed using Unscrambler software  to classify the samples using their explained variables in the following spectral ranges; 3000–2800 cm


Can next-generation sequencing reveal rationale for the dichotomous biological activity of epithelioid hemangioendothelioma (EHE) while illuminating potentially actionable alterations?WWTR1-CAMTA1 fusion, with 18.4% of participants exhibiting a potentially targetable variant. Participants with stage III/IV EHE were more likely to exhibit a secondary pathogenic variant.In a cross-sectional study of next-generation sequencing results collected from 49 participants diagnosed with EHE, more than half of patients with EHE profiled exhibited pathogenic genomic variants in addition to the Next-generation sequencing may identify secondary genomic variants that are associated with EHE aggressiveness; additionally, these variants may represent potential therapeutic targets. This cross-sectional study characterizes secondary genomic alterations in patients with epithelioid hemangioendothelioma and their association with clinical outcomes. WWTR1-CAMTA1 fusion. The clinical course of EHE exhibits a dual nature. The condition is often indolent but can rapidly grow and metastasize unpredictably. No biomarkers to date are available to predict this phenotype. The hypothesis of this study was that better defining the genomic landscape of EHE using next-generation sequencing could offer additional therapies and insight into clinical outcomes.Epithelioid hemangioendothelioma (EHE) is a rare, malignant vascular sarcoma characterized in most cases by a To characterize secondary EHE genomic alterations and their association with clinical outcomes.Multicenter, cross-sectional, retrospective study of next-generation sequencing results collected from participants diagnosed with EHE. Data were abstracted between May 1, 2013, and May 31, 2019. This analysis was conducted from January through June 2019. Summary genomic data were provided by commercial genomic testing companies.Presence or absence of secondary pathogenic genomic variants and their association with disease stage and clinical features.WWTR1-CAMTA1 fusion; 26 participants (57.1%) exhibited a pathogenic genomic variant secondary to the WWTR1-CAMTA1 fusion; and 9 participants (18.4%) exhibited potentially targetable genomic variants. Commonly altered genes included CDKN2A/B, RB1, APC, and FANCA. Participants older than 45 years at diagnosis had an increased prevalence of secondary genomic variants that was not statistically significant  and were more likely to have a clinically targetable variant . In 14 participants with clinical data available, those with stage III/IV EHE were more likely to exhibit a secondary pathogenic genomic variant . Participants with stage III/IV EHE were diagnosed at an older age  and had elevated WWTR1-CAMTA1 fusion expression that was not statistically significant .A total of 49 participants with EHE were assessed for the presence or absence of secondary genomic variants. Of these, 32 (65.3%) were female; the mean (SD) age at diagnosis was 49.9 (18.3) years . In all, 46 participants (93.9%) had confirmed Although EHE exhibits few secondary genomic variants, presence of key secondary variants may be prognostic for aggressive EHE. Further research is needed to confirm this finding and determine whether more intensive upfront treatment is necessary for these patients. Key molecular biomarkers indicative of EHE course have yet to be established5; however, mitotic count and tumor size have been associated with prognosis.6 While limited disease can be amenable to observation or local therapy, metastatic EHE is typically resistant to chemotherapy and carries a poor prognosis. Treatment for advanced-stage EHE is not well established.7 Pathway-specific targeted therapies hold some promise, but improved systemic therapies are still needed.8Epithelioid hemangioendothelioma (EHE) is a rare vascular sarcoma with a prevalence of approximately 1 per 1 000 000 persons.9 In this article, we present the largest assessment, to our knowledge, of the clinicogenomic landscape of WWTR1-CAMTA1 (WC) fusion–associated EHE.Few reports describe the genomic landscape of EHE outside of the driver fusions with their clinical correlates and have described a mostly quiet genome.STREGA) reporting guideline.10Data were abstracted between May 1, 2013, and May 31, 2019. This analysis was conducted from January through June 2019. Summary genomic data was provided by commercial genomic testing companies. This study is reported in accordance with the Strengthening the Reporting of Genetic Association Studies WC fusion were considered secondary variants. Gene enrichment was performed using Superpaths14 (eTable 2 in the 15Pathogenic variants and variants of unknown significance (VUS) were included in our analysis. Genomic variants identified apart from the t tests were used. For categorical variables, χ2 tests were used to generate P values and a test of proportions was used to generate 95% confidence intervals of proportion difference. Continuous data are presented as mean (SD) unless otherwise stated and 2-tailed P values ≤.05 were considered statistically significant.All data were analyzed in R statistical software version 3.4.3  or Prism analysis and graphing software version 8.0.0 (GraphPad). For continuous variables, WC fusion confirmation. These participants were primarily female (29 patients [63.0%]), and the mean (SD) age at diagnosis was 50.2 (18.5) years . Full demographic characteristics are available in the WC expression from available participants demonstrated a right-skewed, log-normal distribution , 46 (93.9%) had WC fusion as a sole pathogenic genomic variant. A single additional pathogenic variant was identified in 14 participants (28.6%), while 2 or more pathogenic variants were identified in an additional 14 participants. The most commonly identified secondary variants were seen in CDKN2A , CDKN2B , RB1 , ATRX , APC , and FANCA   exhibited genomic variants in multiple pathways. Commonly altered genes included CDKN2A/B, RB1, APC, and FANCA. Sex did not segregate secondary genomic variant frequency   exhibited a P = .32) B.P = .02)   C. NotablP = .03)  B.P = .006) (P = .05)  . Participants with stage III/IV EHE were significantly more likely to exhibit a pathogenic secondary genomic variant  A and C. P = .05) B and hadWC fusion–positive EHE. Of the 49 participants included in this study, 46 were positively identified to have the WC fusion with no additional fusions detected. An additional 3 participants with EHE were included after histopathological review. The less common EHE fusion, YAP1-TFE3,16 was not explicitly tested for by the OmniSeq panel. No participants tested with the FMI panel were identified to have concurrent WC and YAP1-TFE3 fusions. We have included this OmniSeq data as well, given the high likelihood of a WC fusion.Epithelioid hemangioendothelioma is characterized molecularly by its primary gene fusions, but owing to the rarity of this disease, little is known regarding the clinical significance of secondary genomic alterations. Here, we present the largest assessment, to our knowledge, of the genomic landscape of CDKN2A/B locus, corresponding to well-studied tumor suppressor genes responsible for regulation of the cell cycle and p53-mediated apoptosis. The data available here are unable to test the importance of CDKN2A/B loss in the natural history or development of EHE. Further study is necessary to identify the role of CDKN2A/B loss in EHE. In other sarcomas, including gastrointestinal stromal tumors, loss of CDKN2A expression is associated with poor prognosis and a greater potential for metastatic disease.19 The biological meaning of CDKN2A/B loss in EHE requires further elucidation.Although half of all EHE tumors included in this study exhibited a pathogenic secondary genomic variant, it was rare for a tumor to have 2 or more secondary variants present. The identified variants are linked to well-studied oncogenic pathways. The most prevalent gene alteration identified in this study was deletion of the WC fusion. Taken together, these data suggest that the fusion event may represent the first step in the development of EHE with a secondary genomic change required for tumor aggressiveness. This has several potential practice implications: for one, participants with newly diagnosed EHE could be considered for genomic profiling to evaluate the presence or absence of secondary alterations; in addition, participants with EHE with secondary alterations may potentially be considered for more aggressive treatment. Prospective clinical trials will need to confirm this guidance.Approximately 20% of EHEs studied exhibited a clinically actionable secondary genomic alteration. Further assessment identified an enriched prevalence in participants aged 45 years or older at diagnosis. In our clinically enriched subset, stage III/IV EHE was strongly associated with the presence of pathogenic secondary genomic variants and older age. Importantly, this was true when either including or excluding participants lacking confirmation of the To our knowledge, this is the largest genomic assessment of EHE to date; however, limitations inherent to studies of extremely rare diseases apply here. As tumor sequencing is not a standard recommendation in the treatment of EHE, the data available may suffer from selection bias toward more aggressive EHE. Additionally, the 2 next-generation sequencing platforms used to test for genomic variants are targeted to a specific set of genes and vary significantly in their coverage. Whole-genome sequencing approaches may provide a more comprehensive assessment; however, the targeted panels used here provide strong, validated assessment of genes known to have biological and clinical associations with cancer. It is important to note that participants who underwent sequencing by FMI and OmniSeq exhibited similar trends in association between genomic variants and tumor stage. Secondary genomic variants are certainly important in EHE; however, the prevalence of these alterations may be lower in a prospectively curated data set. Additionally, this data set is limited temporally and is unable to differentiate between passenger and active genomic variants. Further longitudinal research is necessary to define the genomic progression of EHE.WWTR1-CAMTA1 fusion exhibited a secondary genomic variant, with up to 20% that are potentially clinically actionable. Participants with advanced-stage EHE were significantly more likely to have secondary genomic variants. Prospective, multigroup clinical trials are necessary to confirm these findings and their clinical utility.In this study, more than half of participants with EHE and


Clinical and imaging follow-up coupled with cerebrospinal fluid (CSF) and possibly serum profiling could provide information on disease activity and disability evolution in multiple sclerosis patients.We describe the case of a relapsing-remitting MS patient whose history was characterized by failure of several therapeutic approaches and sustained disease activity. By using a highly sensitive immunoassay methodology, we examined protein expression of 70 inflammatory/cytotoxic molecules in two consecutive paired CSF and serum samples, obtained respectively in 2006 and 2013. At disease diagnosis, elevated CSF protein levels of an inflammatory pattern, including CXCL13, CXCL12, IFNγ, TNF, sTNFR1, IL8, sCD163, APRIL, BAFF, pentraxin III and MMP2 were found compared with a group of controls. At the second lumbar puncture, sustained disease activity was accompanied by considerable (more than 2 fold changes) increase expression of most of these inflammatory molecules while no significant changes in serum inflammatory markers were detected in the two consecutive serum samples.Elevated CSF protein expression of pro-inflammatory mediators, possibly specifically associated to GM demyelination, could remain stable or increase over time in patients with active multiple sclerosis. We underline the role of fluid analysis in understanding the pathophysiology of the disease and providing information on possible markers of disease activity and evolution.The online version of this article (10.1186/s12883-019-1455-7) contains supplementary material, which is available to authorized users. Early identification of the specific immune-phenotype of each multiple sclerosis (MS) patient would be particularly important at a stage in which the most appropriate disease modifying agents could be early provided, in order to improve outcomes before the onset of irreversible tissue damage . Up to nWe here describe a high and increasing over-time inflammatory CSF profile associated with sustained clinical and MRI disease activity in a MS patient.A 31-year-old woman was diagnosed with relapsing-remitting multiple sclerosis (RRMS) in December 2006 , according to revised McDonald criteria  after an3), of which 6 probably intracortical, were detected. After a new optical relapse with multiple active brain lesions, plasma exchange therapy allowed recovery of visual impairment.From April to August 2013 (time of second LP) she was treated with Natalizumab, stopped due to a relapse with atypical brain MRI lesions. CSF examination, repeated for suspected brain infection, excluded viral or opportunistic infections; neither OCB or increased intrathecal Ig synthesis were detected. Non conventional 3 T MRI analysis, with DIR sequences, showed significant cortical/iuxtacortical lesion load  which she carried on until March 2015. Thereafter for 3 months she was given oral dimethyl-fumarate, without effects in reducing disease activity  in protein levels in the two CSF samples collected at t0 and t1, we found that at time of diagnosis 42 inflammatory molecules overexpressed  in CSF of our patient  after 7 years  was used to assess the presence and levels of 70 inflammatory/cytotoxic proteins in paired serum and CSF samples obtained from the examined MS case at the time of diagnosis 2006, t0) and in 2013 (t1), following the procedures previously optimized . By usin006, t0 aOn the contrary, no substantial changes in levels of the same inflammatory proteins have been observed in the two consecutive paired serum samples and when comparing these with a pool of control sera (data not shown).In addition, by using ELISA procedure previously optimized for the analysis of CSF protein expression of neurofilament light chains (Nf-L) , we founIn this report, we have described the concurrence of clinical and MRI activity with the increase over time of specific CSF inflammatory markers.Our data supports the hypothesis that an intrathecal inflammatory profile, including in particular CXCL13, IFNγ, TNF, CXCL12, LIGHT, IL6 and IL10, present at the time of diagnosis, could persist and characterize all over the disease evolution, being not easily targeted by different treatments administered. Together with increased Nf-L CSF levels over time, characterization of this profile could help the identification of prognostic inflammatory and neurodegenerative biomarkers of disease phenotypes and evolution . InteresAmong the molecular changes observed in the CSF during the follow-up period Fig. , the incAdditional file 1:Table S1. Values of evaluated CSF proteins in the two consecutive samples obtained respectively in 2006 (t0) and 2013 (t1). Double arrows indicate significant changes (p < 0.05), measured by non-parametric Mann-Whitney test. Concentrations of the molecules are expressed as pg/ml/mg. (DOCX 33 kb)


The number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics. However, digital PCR (dPCR) offers several advantages over qPCR, making this new technique appealing also for GMO analysis. This critical review focuses on the use of dPCR for the purpose of GMO quantification and addresses parameters which are important for achieving accurate and reliable results, such as the quality and purity of DNA and reaction optimization. Three critical factors are explored and discussed in more depth: correct classification of partitions as positive, correctly determined partition volume, and dilution factor. This review could serve as a guide for all laboratories implementing dPCR. Most of the parameters discussed are applicable to fields other than purely GMO testing. Genetically modified organisms (GMOs) have already passed a two-decade milestone of presence on the market, and the number of GMOs as well as the worldwide area planted with biotech crops continues to increase steadily . Many coThe most commonly used protein-based methods for detection and quantification of GMOs are enzyme-linked immunosorbent assay and use of lateral flow devices  . AlthougPolymerase chain reaction (PCR) is the leading DNA-based method for GMO detection. A second generation of PCR—that is, real-time quantitative PCR (qPCR)—has been the gold standard for GMO trait detection and quantification for more than a decade. The PCR-based GMO detection analysis approach usually starts with qualitative screening for the presence of genetic elements commonly found in GMOs, such as cauliflower mosaic virus 35S promoter (p35S) and nopaline synthase terminator (tNOS) , 13. QuaIn terms of qualitative PCR assays, prespotted plates have been effectively used for detection of multiple GMOs in various crop plants , 17. TheThere are other DNA-based detection methods, which also provide a multiplex detection system, such as capillary gel electrophoresis and different kinds of microarrays , but thLoop-mediated isothermal amplification (LAMP) is a simple qualitative detection method which provides results in less than half an hour , 25. OneAnother technology in the spotlight is next-generation sequencing (NGS). This technology is an alternative method for detection of authorized and unauthorized GMOs in food and feed chains –32. TherDigital PCR (dPCR) technology emerged as a third-generation PCR technique. It was first described as a concept in the 1990s , 37 withIn comparison with conventional end-point PCR and qPCR, dPCR has a number of advantages. The biggest advantage is the capacity of dPCR for absolute quantification of a target without reference to a standard/calibration curve. This minimizes the effect of matrix differences between the calibrant and the test sample, which could cause different amplification efficiencies –51. BecaSeveral dPCR platforms are available Table  and geneIn a cdPCR, the reaction is divided into hundreds or thousands of chambers on a single plate or array. The first one that was available, Fluidigm’s Biomark HD, has already been shown to be suitable for GMO analysis . ReportsA combination of droplet- and chip-based technology platforms is provided by Stilla’s Naica system for crystal dPCR. It uses a microfluidic sapphire chip, with integration of droplet formation, amplification and readout in a single consumable. It also has three-colour detection, enhancing the options for multiplex dPCR . The NaiOverall, the currently available dPCR systems are relatively diverse, but nevertheless they are all theoretically suitable for absolute quantification of GMOs. An attempt has already been made to compare different platforms side-by-side , and theDNA quality is a key factor for successful PCR. The type of samples used, the DNA extraction methods, etc. can affect the quality of extracted DNA (e.g. presence/absence of inhibitors), which can have an impact on amplification with PCR . Demeke As mentioned already, dPCR assays have been reported to be less sensitive to inhibitors compared with qPCR , 65–67. Usually manufacturers of dPCR equipment recommend restriction digestion or fragmentation of DNA samples before dPCR assay. This allows separation of possible tandem gene copies and can reduce the sample viscosity and improve template accessibility. Enzymatic digestion of DNA should be carefully planned to avoid any damage in the amplicon region. It is recommended to perform analysis on digested and non-digested DNA samples at the beginning to see the effect on the final quantification. Such an approach was reported for MON810 maize DNA, and it was shown that for the purpose of GMO quantification enzyme digestion was not necessary . Other fThe amount of DNA used for dPCR can differ according to the instrument used and the sensitivity required. For example, up to 1000 ng DNA can be used for RainDrop ddPCR to detect a low concentration of genetically modified materials , and 100The manufacturer-recommended concentrations of 250 and 900 nM for the probes and primers, respectively, usually perform well for ddPCR assays. However, optimization can in some cases reduce the concentrations to limit the amount of primers and probes used. Nevertheless, more thorough optimization of primer and probe concentrations is especially important for multiplex dPCR assays. Probes are generally labelled with FAM, HEX or VIC, and non-fluorescent black hole quenchers are generally used. If multiplexing is performed in one fluorescent channel, the concentration of probes and/or primers should be optimized thoroughly to allow clear separation of clusters . It is aFor PCR, an annealing temperature of 60 °C is generally used. The annealing temperature has an effect on the resolution between clusters of positive and negative partitions (resolution is increased by lowering of the temperature). However, one must be extremely careful with lowering the annealing temperature as non-specific products can be amplified . LuckilyR2 values may not necessarily show suitability for PCR; thus, they suggested a robust weighted least squares approach as a suitable alternative [The performance of an assay for the purpose of GMO detection and quantification must, at least in the EU, be characterized and needs to comply with the minimum performance parameters . Parameternative .One of the most crucial steps in Bio-Rad’s ddPCR workflow is the transfer of the fragile freshly generated droplets from cartridges to the PCR plate. According to the manufacturer’s recommendations, the transfer of generated droplets with a constant low pipetting speed (suction) and an appropriately steep angle of filter tips gently touching the microtitre plate wall helps to minimize mechanical disruption of the droplets. The amount of accepted droplets can increase with practice and pipetting optimization , but perTc is the mean target concentration (copies per microlitre), P is the number of positive partitions, and R is the number of partitions analysed. As can be seen from Eq. Vd). A dilution factor for the original sample before PCR (D) is also considered in the equation. Essentially, three factors affect the final result: (1) correct classification of partitions as positive, (2) correctly determined partition volume and (3) a dilution factor.In principle, as already mentioned, the absolute target concentration in a sample is calculated on the basis of number of positive partitions and all accepted partitions and by use of a Poisson distribution . The finFluorescence readout is performed for most dPCR platforms with a dedicated machine after PCR or the fluorescence of partitions is measured in each amplification cycle. Finally, the raw result is the fluorescence amplitude of each individual partition. Digital PCR (dPCR) providers offer software for visualization of fluorescence readout; however, raw results can also be analysed independently with use of other tools (e.g. R ). ClassiTo facilitate more reliable classification of partitions, the assay must be optimized in a way that there is as low number of partitions with intermediate fluorescence as possible and that the resolution between the positive and the negative cluster is at least 2 . NeverthThe partition volume is one of the most critical factors affecting target absolute concentration calculation. Discrepancies between partition volumes assigned by the manufacturer and measured in independent laboratories have been reported for ddPCR platforms , 80–82. Accurate dPCR quantification is important in clinical and diagnostic decisions, and thus it is important to at least consider the possibility of incorrect volume and implement this in the calculation of expanded measurement uncertainty. However, in the GMO testing field, the final result is a relative genetically modified content, with the ratio of transgene to endogene, and as such an incorrect droplet volume would affect the absolute copy number of the transgene and the endogene in the same fashion, and thus the relative ratio would not be affected.In dPCR, samples are usually diluted before the reaction because of the limited dynamic range. The target concentration in the stock material is then calculated with use of dilution factors, factors by which the sample was diluted before dPCR. To minimize uncertainty due to pipetting and not to compromise the accurate absolute measurement of copies by application of an incorrect dilution factor, the best approach is to perform all pipetting steps, including preparation of DNA dilutions and reaction mixtures, gravimetrically, with use of calibrated mass balance. This approach was used in studies with the purpose of showing accuracy of measurements with dPCR and to minimize the measurement uncertainty , 80, 84.http://www.decathlon-project.eu). The results showed good comparability of determined values between laboratories in terms of absolute copy numbers determined for each target and GMO content . Some have the option of even higher multiplexing, because of the availability of filters for up to five fluorescence channels Table . Duplex nt Table .Table 3AThe choice of the reporters for TaqMan probes is limited by the availability of only a few fluorescence detection channels . Therefore, a higher level of multiplexing in dPCR can be achieved by other approaches, summarized by Whale et al. : amplituDigital PCR (dPCR) is being used for a wide range of applications in medical, environmental and agricultural areas. The most obvious advantage of dPCR is the possibility to obtain accurate absolute target concentration with no standard curve requirements. Selection of the proper dPCR instrument for a particular laboratory’s need is important. Currently available dPCR instruments seem to be suitable for quantification of GMO events. However, there is variation in throughput and sensitivity, and thus laboratories should assess their needs and budget before making a final decision. Assays used for qPCR can readily be transferred to dPCR; nevertheless, some optimization of the primer and probe concentrations might potentially improve the overall assay performance. A more thorough evaluation and/or verification is essential, especially for assays used in a multiplex dPCR format. Many reports have shown that dPCR is less sensitive to inhibitors compared with qPCR, indicating that it might be a method of choice for samples where the presence of inhibitors is expected. It has also been reported that the DNA extraction method used and DNA quality affect dPCR results. Care must also be taken with partition or droplet volumes assigned by the manufacturer, as the actual values could differ and adjustments may be necessary. As the final GMO content is presented as a relative value, some factors which affect absolute quantification can be partially ignored if they affect both the target and the endogene in the same way. The advantage of using absolute quantification of GMOs (transgene and endogene) is in the elimination of the need for a standard curve generated from certified reference materials. Cost-efficiency is currently still on the qPCR side for simplex assays; however, multiplex assays shift the cost-efficiency towards dPCR.Digital PCR (dPCR) has the potential to replace real-time qPCR. Further research on evaluation of different dPCR instruments and collaborative studies to confirm the wide applicability of the system will thus be useful. Another challenge is the inability to detect unauthorized GMOs with current DNA-based technologies. Genome-edited plants are also gaining popularity. Genome editing allows the introduction of insertions, deletions and substitutions at predetermined sites in the plant genome with use of designer nucleases (e.g. CRISPR/Cas9) . Althoug