Neuroendocrine neoplasms (NENs) are relatively rare neoplasms occurring predominantly in the gastrointestinal tract and pancreas. Their heterogeneity poses challenges for diagnosis and treatment. There is a paucity of markers for characterisation of NEN tumours. For routine diagnosis, immunohistochemistry of the NEN-specific markers CgA and synaptophysin and the proliferation marker Ki-67 are used. These parameters, however, are qualitative and lack the capacity to fully define the tumour phenotype. Molecules of epithelial–mesenchymal transition (EMT) are potential candidates for improved tumour characterisation. Using qRT-PCR, we measured mRNA levels of 27 tumour markers, including 25 EMT-associated markers, in tumour tissue and matched non-tumour tissues for 13 patients with pancreatic NENs. Tissue from patients with three different grades of tumour had distinctly different mRNA profiles. Of the 25 EMT-associated markers analysed, 17 were higher in G3 tissue relative to matched non-tumour tissue, including CD14, CD24, CD31, CD44, CD45, CD56, CK6, CK7, CK13, CK20, NSE, CDX2, CgA, DAXX, PCNA, laminin and Ki-67. The differences in levels of seven EMT-associated markers, Ki-67, DAXX, CD24, CD44, vimentin, laminin and PDX1 plus CgA and NSE (neuroendocrine markers) enabled a distinct molecular signature for each tumour grade to be generated. EMT molecules differentially expressed in three tumour grades have potential for use in tumour stratification and prognostication and as therapeutic targets for treatment of neuroendocrine cancers, following validation with additional samples. Neuroendocrine neoplasms (NENs) are a relatively rare, heterogeneous family of tumours that occur mainly in the gastrointestinal tract 62–67%) and lung (22–27%), with 7% located in the pancreas ,2. Tumou–67% and Routine diagnosis of NENs is based on tissue biopsy of chromogranin A (CgA), a protein found exclusively in the secretary granules of endocrine and neuroendocrine cells ,8, and s2 and Ki-67 < 3% , Grade 2 (intermediate) with a mitotic rate of 2–20 mitoses/2 mm2 and Ki-67 of 3–20%  and Grade 3 (high) with mitotic rates of >20 mitoses/2 mm2 and Ki-67 > 20%  [NENs are graded based on the mitotic index using Ki-67, a marker for proliferation, and using immunohistochemistry (IHC) as a diagnostic tool. Ki-67 is a nuclear antigen present at all stages of the cell cycle except the G0 phase . It was NET, G3) . NeuroenAlthough Ki67, CgA and synaptophysin have been used for regular diagnosis of NETs, there have been studies questioning the reliability of these markers and the basis of their use for treatment decisions. Inter- and intra-observer variability in the counting of Ki-67 has been noted, with a caution to pathologists to be aware while grading . A majorTo date, pathological reports of NETs have been based on the use of a small number of markers using IHC and only on tumour tissue without taking into account matched non-tumour tissue. The paucity of markers in NET management has remained a significant problem. The New Multinational and Multidisciplinary Delphi Consensus meeting of a team of NEN experts concluded the need for novel makers as one of the key gaps that needs to be addressed . This caThe epithelial–mesenchymal transition (EMT) is a collection of cellular changes that are a critical during development. Inappropriate activation of the EMT in cancer accounts for tumour dissemination from the primary to secondary sites . It provIn a previous study, to improve the definition of tumour phenotypes, we tested a panel of 23 tumour-associated markers to profile matched tumour tissue relative to non-tumour tissue from a patient with a G1 pancreatic neuroendocrine tumour . We utilIn the current study, we measured mRNA levels of the panel of 11 EMT-associated markers in tissue from patients with grade 1, 2 and 3 pancreatic neuroendocrine tumours (pNETs) and generated a molecular profile specific to each tumour grade.mRNA levels of 27 tumour-associated markers were measured in tumour tissue relative to non-tumour in all 13 patients grouped into Grade 1, Grade 2 and Grade 3 . RelativThe outlier samples that did not fit in the overall pattern seen within each grade included CgA and CD56 in Grade 1 which were lower in Patient 1 relative to that in non-tumour tissue, while the other G1 tissues were higher in the tumour tissues relative to those in the non-tumour tissue. Within G2, Ki-67 was lower in Patient 2, and CD24, CDX2 and CD45 were higher in Patient 1 relative to those in the non-tumour tissue, in contrast to the other tissues. In Patients 1 and 3, CD44 and CD49 were high in two samples and lower in the remaining three tissues, relative to those in the non-tumour tissue. In G3 samples, CD24 and laminin were lower in Patient 4 and in contrast to the other G3 samples, while vimentin was higher in Patient 3, ECAD higher in Patient 2 and CK20 higher in Patient 4 relative to those in the non-tumour tissue and in contrast to the other G3 samples.As the majority of markers within each grade showed consistent trends, the average mRNA marker levels were determined. In G1, the mRNA levels of CD56, CgA, ECAD, P53, PDX1 and Ki-67 were higher in tumour tissue relative to those in non-tumour tissue and CDX2, DAXX and laminin showed lower expression levels in tumour tissue A. In G2,As most reports present tumour data without matching non-tumour controls, we compared mRNA marker levels in tumour tissue only, between different grades . The mRNAmong the EMT markers, vimentin was most highly expressed in G2 relative to that in G1 tissue E. b-cateThe markers of differentiation CDX2 and CD14 were the lowest in G3 with no differences between G1 and G2. PDX1 was lower in both G2 and G3 relative to that in G1, and for CK6, CK7, CK13 and CK20, no differences were seen between grades G.The tumour suppressor marker p53 was the highest in G2, and no differences between G1 and G3 were observed H.Immunohistochemical analysis indicated that CgA expression was absent in non-tumour tissue, while stained cells represented 25–50% in G1, less than 25% in G2 and 25–50% in G3 , CD44 (G2), DAXX  and CK7 (G3) did not correlate with the protein expression. These data are consistent with our previous study comparing mRNA and protein markers in a G1 pancreatic NET sample which showed that CD44 and DAXX mRNA and protein levels in G1 did not correlate . Poor comRNA levels of some markers within grades did not show significance and for these, correlation with the protein could not be determined. A larger cohort of samples would aid in achieving statistical significance.The patterns of expression of mRNA within each grade may provide insights into the metastatic status of tumours. Based on the pathology report, the tumour sample of patient G2.1 was well-differentiated and non-metastatic . The marThe EMT markers that we analysed showed variations between grades that were generally consistent with the known roles of these molecules in the EMT process underlying cancer progression. The E-cadherin-catenin complex has a dual function in cell/cell adhesion and in transcription. The loss of the E-cadherin-catenin complex from cell junctions is a major hallmark of metastatic progression and EMT ,33. NeurVimentin expression is linked to migration and tumour cells metastasis . VimentiCD31 is an endothelial marker present in both normal and patient blood ,43. EndoTumourigenesis is accompanied by cellular changes in different categories of EMT markers. Thus, we grouped the markers according to their categories, including proliferation, cell adhesion, altered cytoskeleton, cell migration and metastasis, cellular de-differentiation and tumour suppression to determine how they varied across Grades 2 and 3 relative to a G1 tumour as a baseline. It excluded the relative mRNA changes between tumour and non-tumour tissue. The markers that distinguished G3 tumours from the lower grades were higher levels of synaptophysin, CD56, Ki-67, CD24, CD44, β-catenin and E-cadherin. Markers that were lower in G3 compared to G1 and G2 included CgA, CD31, CD45, vimentin, laminin, CD14 and CDX2. The markers that distinguish G2 tumours from G1 or G3 included higher expression of CD31, vimentin, CD44, laminin and p53 and lower expression of CD56, CD24, PCAD and PDX1.The nine most relevant markers from each category can be visualised in the form of a molecular signature that indicates the profile differences of between the grades. The nine markers include CgA, NSE (NE origin), Ki-67, DAXX (proliferation), CD24, CD44 (stem cell), vimentin (EMT), laminin (cell adhesion) and PDX1 (differentiation). Two of these are neuroendocrine-specific markers, while seven are EMT-related. This collection constitutes a biomarker panel for neuroendocrine tumours indicating tumour progression from Grade 1 to Grade 3. It demonstrates the link between tumour progression and validates the link between expression of EMT markers and tumour progression. The utility of biomarker panels has previously been shown in breast cancer to predict response to taxane-based neoadjuvant chemotherapy  and in tThe main limitation of the study was the limited number of samples. Nevertheless, with a relatively small sample size, consistent patterns in the expression of biomarkers were obtained that enabled characterisation of tumour phenotypes between grades.Pancreatic tumour specimens with corresponding matched adjacent non-tumour tissue from thirteen patients with pancreatic neoplasms including four G1, five G2 and four G3 samples were obtained from the Kolling Institute Tumour Bank and the Victorian Cancer Biobank and stored at −80 °C until use. Regions of tissue showing normal pancreatic morphology were classed as non-tumour, and regions where the tissue was disorganised, containing cells with irregular nuclei and heterogeneity of size, were classified as tumour tissue . Table 3Twenty-seven markers that correlated with a range of tumour-associated characteristics that had been previously tested in a pancreatic tumour case study  were selThe experimental protocol received an ethics approval from Deakin University STEC-09-2018 ACKLAND.A 2 mm × 2 mm frozen tissue was cut and ground using a mortar and pestle in the presence of liquid nitrogen and further homogenised by passing through a 20-gauge needle and syringe multiple times. RNA was extracted from the tissue samples using an RNeasy mini kit (Qiagen) as per the manufacturer’s instruction. RNA concentrations were measured using a Thermo Scientific NanoDrop 1000. Only RNA that passed the QC of 260/280 ratio of 2 was reverse transcribed into cDNA.RNA was converted into cDNA using an Applied Biosystems High-Capacity cDNA Reverse Transcription Kit as per the manufacturer’s instructions. A working solution of 5 ng/uL was prepared by diluting the stock using nuclease-free water.−ΔΔCt formula. mRNA transcript levels of each marker were measured in tumour tissue relative to matched non-tumour tissue. The relative expression of markers was first expressed as fold change calculated using b-actin as the endogenous control. Tumour marker mRNA levels were then expressed relative to the control non-tumour tissue which was given an mRNA expression value of one.Ten milliliters of PowerUp SYBR Green Master Mix (Applied Biosystems), 5 ng of cDNA template, 250 nM of each primer was made up to 20 mL in a PCR reaction/well using nuclease-free water. A template (20 ng) was used for markers which were expressed in very low levels. GAPDH and b-actin were used as the endogenous control. Each run also included negative controls of a master mix without cDNA to rule out contamination. The PCR cycle was 50 °C—2 min, 95 °C—10 min and 45 cycles of 95 °C—15 s, 60 °C—1 min and a final extension for 95 °C—15 s. The plate was loaded onto a Bio-Rad CFX96 qPCR machine, and the marker levels were analysed using the relative quantification method (ΔΔCt). All markers were tested for 3 individual qPCR runs, and triplicate experiments were run for each sample. The fold change was calculated using the 2Tissue Sectioning: Tissue samples of size 3 mm × 3 mm were cut and frozen in liquid nitrogen in OCT. Sections of 5–8 mm thickness were sectioned and collected on a charged slide using a Thermo Scientific HM525NX cryostat. The slides were air-dried and stored at −80 °C until use.Frozen sections were rinsed three times with 1× PBS for 10 min then fixed in 4% paraformaldehyde (PFA). The slides were washed three times with 1X PBS then incubated in 0.3% hydrogen peroxide for 10 min followed by 10 min incubation with 0.1% Triton × with three 1× PBS washes between the two incubations. The sections were blocked in 2% BSA for 45 min and then incubated with primary antibodies at 4 °C overnight. The following day, the slides were washed using 1× PBS and incubated with horseradish peroxidase or alkaline phosphatase enzyme tagged secondary antibody for 30 min. The corresponding substrate, ImmPACT DAB or alkaline phosphatase (Vector Laboratories) was then added, followed by counterstaining with methyl green or haematoxylin and mounting using a DPX solution (Sigma Aldrich).Images were taken at 40×, 100× and 200× magnifications using an Olympus BX43 light microscope fitted with an Olympus DP22 camera. Images were collected using DP software and analysed by comparing the intensity of the label in the tumour sample to that of the matched non-tumour tissue.t-test with unequal variance was used to find the probability value  between each normal and tumour sample and its positive and negative standard deviation values. p-values less than 0.05 were considered statistically significant. The average of the relative fold change and standard deviation of the samples from the same grade were used to calculate the marker expression profile for each grade.A two-tailed p-value < 0.05 were considered significant. “a” and “b” represent markers expressed significantly differently compared to G1 and G2.Marker expression was studied across tumour grades using G1 as the reference sample. One-way ANOVA with post-hoc Tukey’s test was used, and markers with a Tissue from patients with Grade 1, 2 and 3 tumours had distinct profiles seen in the expression of a range of EMT markers. Most EMT markers had consistently higher mRNA levels in tumour tissue relative to those in non-tumour tissue in all three grades, with expression levels increasing with higher grade. The differences in tumour markers between grades confirmed the link between EMT and tumourigenesis and provided insights into the molecular changes associated with cancer progression. The marker profiles can be visualised as a molecular signature of nine markers, CgA, NSE (NE origin), Ki-67, DAXX (proliferation), CD24, CD44 (stem cell), vimentin , laminin (cell adhesion) and PDX1 (differentiation), that have potential for use in tumour stratification and for identification of clinically relevant subtypes. The value of such a molecular signature needs to be tested in a larger cohort of patients.


Thirst significantly increased from pre- to post-run regardless of treatment (p < 0.001); however, there was no significant difference between the groups (p = 0.492). Feelings of fullness did not change pre-post trial (p = 0.304) or between trials (p = 0.958). Increased fluid availability allows for increased fluid consumption without the negative experience of GI discomfort. Fluid availability may alter drinking behavior; however, it is currently unknown if the availability of fluid impacts behavior and gastrointestinal issues (GI) that are often associated with increased fluid intake. The purpose of this study was to determine if ad libitum (AL) versus periodic (PER) fluid intake influences fluid consumption and GI distress during exercise in trained athletes. Male and female Division I NCAA Cross Country athletes  participated in this counterbalanced crossover study. Each participant completed a moderate intensity 10 km run on two separate occasions. In one trial, participants had unlimited availability to fluid to consume AL. In the other trial, participants consumed PER fluid at stations placed every 3.2 km. Assurance of euhydration prior to each trial was confirmed via urine specific gravity (USG) and urine color. Subjective perceptions of thirst and gastric fullness were assessed pre- and post-exercise via Likert questioning and a visual analog scale, respectively. Participants started each trial euhydrated . Fluid volume consumption was significantly higher during the AL condition compared to PER ( Hydration and fluid balance have been repeatedly shown to alter cognitive and physical performance in general and athletic populations alike ,2. EuhydMaintenance of a euhydrated state during aerobic exercise, even in the absence of thirst, has been previously linked to optimal performance . For exaRegardless of the fluid intake method, fluid availability may strongly influence the fluid intake of exercising individuals . Aragón-While research has established that fluid consumption behavior is multi-faceted, it has not addressed how the availability of fluid may impact fluid intake during exercise . AlthougA group of 11  non-heat-acclimatized NCAA Division 1 Cross Country athletes participated in this study . Participants were assumed to be non-heat-acclimatized due to the time of year that the study took place (February–March) in the United States. To ensure the health and safety of the participants, a health history and Physical Activity Readiness Questionnaire (PARQ) was completed prior to involvement. All participants met the ACSM guidelines for being physically active (at least 3 days per week of at least 30 min of moderate intensity activity for at least 3 months), had not experienced a lower-body injury within the past 6 months from the date of the first visit, nor did they indicate cardiovascular, metabolic, or renal disease. For each visit, participants were asked to refrain from consuming alcohol, caffeine, nicotine, and pre-/post-workout supplements for 12 h prior to each visit, and to avoid strenuous exercising for at least 24 h prior to the time of each scheduled visit. The experimental protocol was approved by the Samford University Review Board (#EXPD-HP-21-S-1) and participants provided their written informed consent prior to participation, in accordance with the Declaration of Helsinki. In a randomized, counterbalanced, crossover study design, participants completed two trials: (1) 10 k run with readily available fluid (AL) and (2) 10 k run with pre-placed fluid (PER) . Prior tIn one trial, participants were given a fluid bottle filled with water to carry with them throughout the duration of the run (AL). To ensure participants always had availability of fluid, additional fluid bottles were placed throughout the course for the participants to use if their initial bottle had become low on fluid. In the other trial, participants were able to consume fluid at fluid stations placed every 3.2 km throughout the course (PER). Participants were given the option to grab a fluid bottle filled with water at the stations and consume fluid for a maximum of 200 m from the fluid station while running. For all trials, participants were asked to confirm their adherence to the instructed pre-trial fluid intake protocol and major deviations from the protocol resulted in the trial being rescheduled. Upon arrival at all trials, hydration was assessed through urine analysis. Participants provided a urine sample upon arrival and urine specific gravity (USG) was assessed using a hand-held refractometer  and urine color was assessed through comparison with a urine color chart . Euhydration was defined as a USG of less than 1.020 . If USG Before beginning and immediately following exercise, nude body mass , thirst, and rating of perceived exertion were assessed. To assess nude body mass, participants entered a private and locked room where they removed all clothing and dried off with a towel (after exercise) to obtain a body mass. Thirst was assessed by asking the participants to rate their sensation of thirst on a 9-point Likert scale with 1 indicating “Not thirsty at all” and 9 indicating “Very, Very Thirsty” . AdditioRate of perceived exertion was recorded according to a Borg scale (6–20). Average heart rate and total duration were monitored in both trials using wrist-worn GPS and heart rate devices . The trials took place over three different days at the same time of day (1500–1700) to accommodate the participants’ schedules. Environmental conditions were assessed at the beginning of each trial using the Kestrel 3000HS Heat Stress Meter .t-tests were used to compare hydration indices  and HR between trials. Two-way repeated measures ANOVAs  were used to assess perceptual measures. Tukey correction was used for post hoc analyses. Alpha was set at p ≤ 0.05, a priori. Data were reported as mean ± standard deviation, Cohen’s d effect sizes (ES) for t-tests, and partial eta squared effect sizes (ηp2) for ANOVAs. Cohen’s d was interpreted according to the following thresholds: <0.2 = trivial, 0.2–0.6 = small, 0.7–1.1 = moderate, 1.2–2.0 = large, and >2.0 = very large [2 was interpreted according to the following thresholds: 0.01 = small effect, 0.06 = medium effect, and 0.14 = large effect [All statistical analyses were completed using Jamovi (The jamovi project 2020) Jamovi (Version1.2)). Paired samples ry large . ηp2 was20 Jamovip = 0.05; ES = 0.67) than the amount of fluid consumed in PER (111.4 ± 110.0 mL).USG, urine color, % body mass loss, HR, and duration were not significantly different between trials . Figure p < 0.001, ηp2 = 0.76; Satiety: p < 0.001, ηp2 = 0.85; Dryness: p < 0.001, ηp2 = 0.79; Taste: p = 0.03, ηp2 = 0.41). Thirst did not demonstrate differences between trials  and there was no interaction effect . Satiety did not demonstrate differences between trials  and there was no interaction effect . Dryness did not demonstrate differences between trials  and there was no interaction effect . Taste did not demonstrate differences between trials  and there was no interaction effect . There was no difference between time , trial , or an interaction effect for fullness . There was no difference between time  for nausea and there was a significant difference between trial . However, there was no significant interaction for nausea .The descriptive results from the visual analog thirst scale are recorded in p < 0.001; PER-pre = 6 ± 2, PER-post = 13 ± 2, p < 0.001, ηp2 = 0.92); however, there were no differences between trials  and there was no interaction effect . Thirst from the 1–9 scale was significantly higher from pre-post trial in both trials ; however there were no differences between trials  and there was no interaction effect . RPE was significantly higher from pre-post trial in both trials (AL-pre = 6 ± 1, AL-post = 12 ± 2, The objective of this study was to determine the impact of fluid availability on fluid intake behavior and perceptual thirst and GI discomfort measures during moderate intensity exercise. The main finding of the current study demonstrates that increasing fluid availability when drinking AL resulted in significantly greater fluid intake over a 10 km run. Importantly, the increase in fluid volume did not result in an increase in GI discomfort, indicating that AL fluid availability may be a practical and ideal hydration strategy during exercise. The literature has established that hypohydration can negatively influence aerobic exercise performance ,21,22. OCurrent results indicate that the amount of fluid consumed had no significant effect on the sensation of thirst or GI discomfort. This is contrary to previous studies that showed a decreased sensation of thirst and increased abdominal discomfort when participants drank more fluid during exercise . HoweverArmstrong et al. summarize thirst behavior into two main categories that affect thirst: homeostatic thirst and non-homeostatic thirst . HomeostAnother factor that may influence fluid consumption behavior is the gastric emptying rate. As fluid is ingested, the pyloric sphincter regulates the rate at which fluid leaves the stomach and enters the rest of the GI tract. The timing and volume of fluid consumption can exceed the stomach’s capacity to distribute fluid, thus, resulting in discomfort and pain. Because of this, even though the sensation of thirst may be present, gastric emptying rates only allow for the consumption of a limited amount of fluid before the stomach feels full . This isThe previous literature has indicated that maintaining a body mass loss <2% is ideal for optimal exercise performance . In the In conclusion, this study provides novel evidence that fluid intake during exercise is influenced by more than just the perception of thirst. Fluid availability is a potent influence of fluid intake behavior and is highly likely to contribute to how and when exercising individuals hydrate. AL fluid intake with readily available fluid resulted in significantly greater fluid consumption compared to fluid intake from stations, without increased GI discomfort. In order to optimize hydration during exercise, individuals should consider carrying a water source with them. This strategy may be particularly impactful when exercising in a hot climate when sweat rates are elevated.


Overall, we demonstrated the potentialof Fc-PepH3 as a versatile platform readily adaptable to diverse drugsof therapeutic value to treat different brain conditions.The frequency ofbrain disease has increased significantly in thepast years. After diagnosis, therapeutic options are usually limited,which demands the development of innovative therapeutic strategies.The use of antibody–drug conjugates (ADCs) is promising buthighly limited by the existence of the blood–brain barrier(BBB). To overcome the impermeability of this barrier, antibody fragmentscan be engineered and conjugated to BBB peptide shuttles (BBBpS),which are capable of brain penetration. Herein, we linked the highlyefficient BBBpS, PepH3, to the IgG fragment crystallizable (Fc) domainusing the streamlined expressed protein ligation (SEPL) method. Withthis strategy, we obtained an Fc-PepH3 scaffold that can carry differentpayloads. Fc-PepH3 was shown to be nontoxic, capable of crossing anin vitro cellular BBB model, and able to bind to the neonatal Fc receptor(FcRn), which is responsible for antibody long half-life ( However, central nervous system (CNS) therapeuticsface a low success rate in disease management, which is somehow relatedto a general poor knowledge of brain physiology, high frequency oftherapeutic adverse events, and absence of certified biomarkers.4 The existence of the blood–brain barrier (BBB) correspondsto an additional difficulty for effective CNS delivery of numeroussmall-molecule and biopharmaceutical drugs. The BBB stands as a selectiveendothelial barrier between the blood and brain compartment that playsa crucial part in brain protection and limits the penetration of therapeuticmolecules.5Brain diseases correspond toalmost 13% of the global health burden, exceeding cardiovascular andcancer diseases.6The brain penetrationof therapeutic molecules is challenged bybarrier properties. In addition, this barrier controls the brain entryof mediators and immune cells. Thus, immune responses within the brainare different compared with other body regions, making the brain animmune privileged site. Hence, treatment of brain conditions remainsa concern.7 The use of immunotherapyand targeted therapies is the most promising. The former consistson stimulating the host immune system to eliminate nefarious cells,9 while targeted therapies rely on the binding of an antibody/antibodyfragment to an epitope at the membrane of cells or mediators.10 The antibody/antibody fragment can be eitherthe therapeutic entity itself, part of a drug-delivery system, suchas a nanoparticle or an antibody–drug conjugate (ADCs), orboth.Various innovative strategiesto treat different brain conditionshave been investigated.11 Otherpharmacokinetic (PK) parameters of antibodies, such as high specificityand long half-life (t1/2), are exploitedby drug developers to deliver potent chemotherapeutic agents thatby their toxicity or poor PK could not be otherwise administered topatients. Upon conjugation to antibodies/antibody fragments, theyshow decreasing off-target effects and/or improved PK.13 Thus, the same strategy can be transposed to other diseases. Unfortunately,CNS-targeted therapies suffer from an important limitation: the brainpenetration of drugs, in general, is hampered by the BBB. Thus, withoutadequate modifications, most therapeutic molecules cannot traversethe BBB and accumulate in the brain, which complicates disease management.Strategies to overcome this limitation and promote BBB antibody crossinginclude the use of antibody fragments and/or their conjugation toBBB peptide shuttles (BBBpS), which are cell-penetrating peptides(CPPs) that are able to specifically cross endothelial cells and accumulatein the brain.14The use of ADCs in cancer treatment has increased in popularityover the years. They display high affinity and tolerability, low drug–druginteractions, and low toxicity.15 followed by others, such as SynB, penetratin, Angiopep-2, and dNP2.17 Our group has contributed with a novel BBBpS, named PepH3, a sevenamino acid peptide derived from Dengue virus capsid (DEN2C),18 a highly basic protein able to translocate cellmembranes.20 In our in vitro BBB models, PepH3demonstrated a translocation above 60%,21 and in vivostudies showed a brain penetration of 0.31% after 5 min injection.18 We further evaluated the translocation capabilitiesof PepH3 upon conjugation to a single domain antibody (sdAb) thatrecognizes β-amyloid peptide 1–42 (bAP42) . After 2 min, a 1.5% (ID/g) brain penetrationof the anti-bAP42 sdAb was observed.22 Thebrain uptake values reported are comparable to values obtained forother peptides (<1.1% ID/g)24 and those of knownCNS drugs .26 Moreover, PepH3 demonstratedselectivity toward endothelial cells compared to other cell linestested (unpublished data), which is a significant advantage sincemost CPPs are widely distributed upon circulation to different tissues.The initial BBBpSdescribed was the human immunodeficiency virustransactivator of transduction (TAT) peptide,t1/2, throughthe neonatal Fc receptor (FcRn) salvage pathway, and increased cytotoxiceffect, owing to its capacity to elicit immune effector mechanisms.27 Indeed, our Fc-PepH3 platform has attractivePK/PD properties with the capability of penetrating the brain. Inaddition, the Fc domain provides a scaffold onto which other moleculescould be conjugated. The result is a drug-delivery platform that improvesthe PK properties and efficiency of drugs and that is capable of BBBtranslocation and brain accumulation  domainof an immunoglobulin (IgG) (MW ±55 kDa) 1. This dmulation 1.t1/2. The first involvedthe chemical conjugation of the synthetic PepH3 peptide to the Fcdomain. Whereas a wide range of chemical methodologies for site-specificconjugation is available,31 not all of them are ideal for the sensitive natureof peptides and/or Fc, as they tend to rely on experimental conditionspotentially detrimental to the species involved . Thus, we selected the streamlinedexpressed protein ligation (SEPL), which is run under physiological-likeconditions,33 and yielded a homogeneous conjugateof high purity. A second challenge was to validate the ability ofPepH3 to facilitate the in vitro BBB translocation of such a largeconjugate. Until now, PepH3 conjugation had involved small payloads.22 Herein, we applied an optimizedin vitro BBB model to show the Fc-PepH3 conjugate translocation ability.Third, we wanted to probe the interaction of the Fc domain with FcRn.Altering the binding affinity to this receptor would compromise someof the hoped-for properties mentioned above. In addition, interactionbetween Fc and FcRn is important for recycling of Fc-PepH3 from thebrain to the blood, which results in a get in/get out homeostasisthat avoids undesirable toxicity associated with accumulation in thebrain.To demonstrate its potential,we addressed the three followingmain challenges throughout the development of the Fc-PepH3 conjugate:(i) generation of Fc-PepH3 conjugates, (ii) validation of the abilityof PepH3 to induce BBB crossing for such a large conjugate, and (iii)ensuring the Fc retains its affinity for the FcRn, responsible forantibody long 34 a new variant of the expressedprotein ligation (EPL) that does not suffer from slow reaction ratesand premature hydrolysis.36 We decided to use SEPLto conjugate the BBBpS PepH3 to the IgG Fc domain . For engineering of the Fc-IntN protein,different ultrafast N-terminal intein fragments might be employed,with Fc-AvaN being the one with the highest rates of Fc-intein fusionsand no effects on Fc glycosylation.34 Tothat end, a previously developed plasmid encoding the Fc-AvaN wasused, and the recombinant protein was produced in a human expressionsystem (Expi293 cells).SEPL requires as building blocksa synthetic thiol-functionalizedlink, usually a Cys-containing peptide  and a recombinantprotein α-thioester. Thus, our first step was N-terminal elongationof the PepH3 sequence with an extra Cys residue and a 6-aminohexanoicacid linker, intended to preserve PepH3 mobility, hence not affectingtranslocation ability. The solid-phase synthesis was performed bythe Fmoc approach in an automated instrument. RP-HPLC/MS confirmedthe identity and purity of the peptide . The hemolyticprofile was also evaluated using freshly isolated red blood cells(RBCs). This standard safety assay is widely applied due to its simplicity,robustness, cheapness, and highly informative nature.38 No hemolytic activity was observed for all antibody fragments.Determining the nontoxic concentration of antibodyfragments wasimportant to establish their potential applicability in an in vivosetting. The in vitro toxicity of antibody fragments and conjugateswas studied on endothelial (HBEC-5i) and human fibroblast (Hs68) celllines. We selected HBEC-5i because it is the endothelial cell lineused in our in vitro BBB model and human fibroblast Hs68 for its wideapplication in preclinical toxicity studies.40 and the choice between the different ones depends on the purposeof the study. In our case, we selected a simple and quick in vitroBBB model optimized in our lab.41 Translocation dataobtained with these in vitro BBB models correlate to biodistributiondata obtained in vivo.18 It consists oftwo chambers  divided by a permeable membrane,which is covered by a monolayer of HBEC-5i cells that in contact witheach other creates tight junctions composed of transmembrane proteins.42Second, we evaluated the translocationcapacity of Fc-PepH3 comparedwith that of other antibody fragments, which were used as controls.Fc-OH is the unconjugated form of Fc-PepH3, lacking PepH3, which intendsto improve BBB translocation, and FC5 was selected based on its well-knowntranslocation properties. In the literature, in vitro BBB models withdifferent levels of complexity are described,Figure S3). The 16.2 ± 4.53% Fc-OH foundin the basolateral compartment at 24 h might be related to a slightlydecreased integrity of the barrier model or to the existence of FcRnreceptors at endothelial cells.43 In anyevent, Fc-PepH3 at 6 h displayed translocation 5.0-fold higher thanthat for Fc-OH, and at 24 h, the percentage of Fc-pepH3 was 2.4-foldhigher than that of Fc-OH. Also, the translocation profile of Fc-PepH3at 24 h was compared with that of recombinant sdAb FC5. FC5 efficientlypenetrated the BBB by receptor-mediated transport (RMT),44 a mechanism different from the absorptive-mediatedtransport (AMT) that drives Fc-PepH3 translocation.21 For FC5, translocation was 4.0 ± 0.56 and 42.0 ± 3.35%at 6 and 24 h, respectively . Furthermore,evaluation of BBB integrity using a 40 kDa dextran conjugated to fluorescein(FD40) revealed that barrier permeability was not compromised by allantibody fragments, excluding a paracellular transport, which validatedthe data obtained .Data obtained for the Fc-PepH3 conjugateshowed it was able totranslocate our model, whereas Fc-OH translocation was significantlylower . These values are in agreement withprevious results,34 thus confirming thatligation was functionally successful.Finally, in order to assess if conjugationhad any effect on Fc t1/2. The conjugate described here provides a first step toward the developmentof a novel therapeutic platform to tackle brain disorders to improvepatient survival and prognosis.Taken together, our resultsdemonstrate the successful developmentof a nontoxic Fc-PepH3 platform, able to transverse an in vitro BBBmodel, using AMT, which is a promising approach that presents someadvantages over RMT. Here, we show that (i) the conjugation of a peptideto an IgG Fc domain using a chemical method is a valid approach forin vivo applications; (ii) the Fc-peptide conjugate is capable ofeffective BBB crossing, to the same extent as a gold standard antibodyfragment (FC5), albeit using a different translocation pathway; (iii)the Fc-peptide conjugate keeps the binding affinity of the Fc domaintoward the FcRn, which is deemed important to prolonging circulationtime and high efficacy; (iv) the Fc-peptide conjugate is nontoxictoward a broad panel of cell lines and erythrocytes, which very likelytranslates to in vivo safety. Using this conjugate, different therapeuticpayloads that could not otherwise be administered due to toxicitycan be delivered, improving PK and decreasing off-target effects.The result would be the generation of novel therapeutics with brainpenetration ability and retain long


We encountered two cases of varicella occurring in newborn infants. Because the time between birth and the onset of the illness was much shorter than the varicella incubation period, the cases suggested that the infection was maternally acquired, despite the fact that neither mother experienced clinical zoster. Thus, we tested the hypothesis that VZV frequently reactivates asymptomatically in late pregnancy. The appearance of DNA-encoding VZV genes in saliva was used as an indicator of reactivation. Saliva was collected from 5 women in the first and 14 women in the third trimesters of pregnancy and analyzed at two different sites, at one using nested PCR and at the other using quantitative PCR (qPCR). No VZV DNA was detected at either site in the saliva of women during the first trimester; however, VZV DNA was detected in the majority of samples of saliva  during the third trimester. These observations suggest that VZV reactivation occurs commonly during the third trimester of pregnancy. It is possible that this phenomenon, which remains in most patients below the clinical threshold, provides an endogenous boost to immunity and, thus, is beneficial. Extreme stress, such as space flight, may cause varicella zoster virus (VZV) to reactivate from latency (in persons who have had varicella or have been vaccinated against it) without producing symptoms. This phenomenon was reported to occur in 50–65% of astronauts during and after short- and long-duration spaceflights ,2 and inWe encountered two newborn infants who developed varicella just after birth, a timeframe that is much shorter than the incubation period for varicella. These observations suggested that VZV was passed from the mother to the fetus prior to delivery. Because neither mother displayed varicella or zoster, it was likely that asymptomatic reactivations of VZV occurred in late pregnancy and provided the virus that completed the varicella incubation period perinatally. Because the varicella in the newborn infants was mild, maternally derived immunity was also transferred and ameliorated the infection. These observations led us to ask whether the asymptomatic reactivation of VZV occurs frequently in pregnancy. To answer this question, we determined whether VZV DNA could be detected in the saliva of a group of pregnant women in the absence of symptoms, as has been reported in astronauts ,2. We alCase 1. This infant was born at Columbia Presbyterian Hospital in New York City in 2018. His mother, a 37-year-old healthy woman presented in active labor at 36 weeks of pregnancy. Her antenatal period was significant only for vaginal candidiasis. She had no history of febrile illnesses or rashes in the antenatal period and no chronic medical conditions. She recalled experiencing chickenpox in childhood and had not experienced clinical zoster. Her serum varicella IgG level was positive prior to pregnancy. Her male baby was delivered by Caesarean section for nonreassuring fetal heart rate with a rupture of membranes at delivery. The APGAR scores were 8 and 9; the infant’s weight was 3295 g. The infant was hypoglycemic and hypothermic shortly after delivery. Ampicillin and gentamicin were administered after blood cultures were obtained. The postnatal examination revealed a mild, diffuse, pustular rash with scattered petechiae. Dermatology was consulted; the differential diagnosis included systemic bacterial or viral infection and transient neonatal pustulosis. On day one of life, bacterial and fungal blood and skin cultures were obtained and were found to be negative. VZV and herpes simplex virus (HSV) cultures from skin lesions were obtained and were found to be negative. Acyclovir 60 mg/kg/dose IV was administered since the infant was thought possibly to have a neonatal HSV infection. Direct fluorescent antigen (DFA) tests from the skin lesions then tested positive for VZV and negative for HSV. The polymerase chain reaction (PCR) tests obtained from skin lesions were positive for VZV and negative for HSV. A diagnosis of varicella was made in the infant, although there was no obvious exposure of the baby to VZV. A lumbar puncture was not performed. The diagnosis of varicella was made in the infant, and treatment with acyclovir was continued for a week. The infant’s rash improved, and the baby appeared entirely well on a follow-up visit.Case 2. This 39-week, 1-day-old male infant was born via Cesarian section due to failure to progress at Pro Medica Russell J. Ebeid Children’s Hospital in Toledo, Ohio, in June 2021. The pregnancy was complicated by gestational hypertension. He presented with vesicular lesions on his right cheek in the first 24 h after birth. Subsequently, a mild rash was noted on his trunk that was thought to be erythema toxicum. Neonatal HSV infection was suspected, but a PCR test on a lesion on the cheek was negative for HSV DNA. It was positive, however, for VZV DNA . As part of routine prenatal testing, his mother was found to have a positive IgG immunoassay for VZV IgG in her first trimester of pregnancy. She recalled experiencing varicella as a child. Except for the rash, the baby appeared well. New lesions developed on day four of life. By day six, all the lesions had crusted, and no further lesions developed. VZV IgG testing was positive on the infant. The mother was asymptomatic throughout the illness of her infant but reported that an aunt who babysat her daughter experienced shingles prior to the delivery. The infant was discharged and went home at 11 days of age.We performed a prospective observational study in order to determine if we could identify evidence of the asymptomatic reactivation of VZV in pregnancy. Pregnant subjects from different trimesters in pregnancy who received prenatal care at our institution were approached and consented to participate in our study. The subjects were recruited from 3 general areas: obstetric ultrasound, the antepartum unit, and the labor and delivery unit. The subjects were excluded if they had any signs or symptoms of COVID-19 infection or other viral illness. Saliva was collected with commercial kits for this purpose obtained from Omnigene-oral Genotek , as described previously . Nested Saliva samples were obtained from 19 pregnant women. These women ranged in age from 25 to 46 years (mean 35). Their ethnic backgrounds were varied and included White (n = 9), Black (n = 4), Hispanic (n = 5), and Asian (n = 1). Ten patients provided a history of having experienced varicella in childhood; three did not, and six could not recall. Three remembered having received the live attenuated varicella vaccine and did not have a history of varicella. Most patients were healthy at baseline; however, two patients had a chronic medical condition: one had cystic fibrosis, and one had systemic lupus erythematosus. Samples were obtained from five women in the first trimester of pregnancy (weeks 9–12), and fourteen from women in the third trimester .The saliva from the patients was independently assayed in two different laboratories: at Columbia and at NASA. From the Columbia laboratory, 11/12 (92%) third-trimester patients were found to have VZV DNA in their saliva by nested PCR. In the NASA laboratory, 7/10 (70%) were positive by real-time PCR. Their viral loads ranged from 508 to 1128 copies/mL with a mean + SE of 717 + 84 copies/mL saliva. Ten saliva samples were tested at both sites; the results agreed in 8/10 samples (80%). Two third-trimester patients had positive saliva samples at Columbia but negative tests at NASA. Saliva from 5/5 women in the first trimester were negative by qPCR at Columbia; 2/2 tested were negative at NASA. At NASA, no reactivation of HSV or cytomegalovirus (CMV) was identified in saliva from nine patients by qPCR.In summary, at Columbia, of 12 women who were studied in weeks 25–40, 11/12 had positive saliva for VZV DNA (92%) using nested PCR. At NASA, of 10 patients studied in weeks 25–40, 7/10 (70%) were positive for VZV DNA by qPCR. In five women whose saliva was studied during the first trimester for VZV DNA, 0/5 were positive .We became interested in whether the reactivation of VZV in pregnancy was common or not after observing two newborn infants with varicella. One reported infant was born at Columbia. The other was encountered 3 years later because the medical team responsible for the care of this infant had heard about the original Columbia patient and brought their patient to the attention of the Columbia physicians. The silent reactivation of VZV is of particular interest because it is a potential stimulant of maternal immunity to VZV. It is interesting that VZV was reactivated in the setting of decreased immunity in pregnancy, which is thought to occur so that the mother does not reject her fetus. Immunity to VZV, however, was not so diminished in the pregnant women that clinical zoster was able to occur. Reactivation was only identified in women during late pregnancy.It is not fully understood why immunity to varicella persists for years, both in individuals who have experienced clinical varicella and also in those who have been vaccinated. Two theories exist ,7: One iThe diagnosis of neonatal varicella in the first infant in this report led us to begin to determine whether the reactivation of VZV could be identified in late pregnancy, when an incipient mother presumably becomes at least somewhat immunocompromised. The second infant was born 3 years later, after the study was underway, at a different hospital. Both cases were demonstrated to be due to VZV by the PCR of skin specimens at Columbia. There is no question about the diagnosis of VZV in these infants. It was, at first, suspected that each baby might have a herpes simplex virus (HSV) infection, but HSV was not detected in either one by PCR, ruling out this possibility. The babies were treated appropriately with acyclovir and recovered. The course of their varicella was mild, as would also be expected, due to the presence in the infants of maternal antibodies to VZV  and antiviral therapy.When these young babies were diagnosed with varicella, it was clear that the VZV had to have come from their mothers, who themselves had no symptoms of VZV infection. The shortest possible incubation period of varicella is 7 days, so postnatal exposure was not possible. The phenomenon of varicella in 1- to 3-day old infants was described previously in three newborns many years ago . In all In the current study, we found that the silent reactivation of VZV during pregnancy was common. This was shown independently in two different laboratories. There was close agreement between the two laboratories for women in the third trimester (92% reactivation at Columbia vs. 70% at NASA). Nested PCR is more sensitive than qPCR, so this result was not unexpected. It is of interest that the saliva samples of five women tested in early pregnancy were negative for reactivation by qPCR at both laboratories.In addition to the rare cases of clinical varicella in neonates who were born to healthy mothers that we described, other data suggest that VZV from healthy mothers can cross the placenta. About 20 years ago, VZV was reported in the cerebrospinal fluid (CSF) of two neonates with neurologic abnormalities and no history of rash. These infants were born to healthy mothers who completed normal pregnancies ,11. It wWe found no evidence in our study on saliva of reactivation of HSV or cytomegalovirus (CMV) in the pregnant women we described. We expected that we might find reactivation of other herpesviruses, such CMV and HSV, which reactivate along with VZV in astronauts , but we About 50 years ago, Hope-Simpson proposed the hypothesis that VZV could reactivate from latency to cause zoster . In addiThe periodic silent reactivation of VZV without symptoms that Hope-Simpson postulated could be a critical factor in the maintenance of long-term immunity to VZV. After primary infection, VZV becomes latent in peripheral neurons, including those of the enteric nervous system (ENS) ,7; moreoSome epidemiological investigators assumed that exogenous exposure to circulating wild-type VZV is critical for the maintenance of immunity to VZV and built computer models to predict what would happen if VZV ceased to circulate and epidemics of varicella no longer occurred . These m


Sepsis-associated endothelial dysfunction and degradation result in release of inflammatory mediators, compromise endothelial permeability, and impair alveolar fluid clearance leading to pulmonary edema. Excessive fluid therapy in septic shock damage the endothelial glycocalyx which will increase capillary leakage. The aim of our study was to assess the relationship of endothelial glycocalyx shedding with hemodynamic and metabolic response to fluid load in patients with septic shock.−1 for 10 minutes. The plasma concentrations of endothelial glycocalyx components including heparan sulfate proteoglycan and syndecan 1 were measured at baseline, 2, 24 hours after fluid load test.Eighteen adult patients were included in prospective observational study. To predict the response to infusion, we performed fluid load test by using crystalloids 7 mL kgrho = 0.48, P  = .04) at baseline and of heparan sulfate proteoglycan with extravascular lung water index  and pulse pressure variation  at 24 hours after fluid load test. The plasma concentration of syndecan 1 correlated with lactate at baseline  and at 24 hours after fluid load test . At 2 hours after fluid load test, the concentration of syndecan 1 correlated with global end-diastolic volume index  in normovolemic patients.We observed associations of syndecan 1 with extravascular lung water index (The shedding of endothelial glycocalyx after fluid load test in septic shock is associated with hemodynamic and metabolic responses and related with the severity of pulmonary edema. The response of endothelial glycocalyx to sepsis depends on fluid therapy with increased glycocalyx shedding after achieving normal volume status of the patient.In septic shock, the concentration of endothelial glycocalyx components correlates with parameters of preload, severity of pulmonary edema, and plasma lactate.The accumulation of extravascular lung water associates with plasma concentration of syndecan 1 during early phase of septic shock, and with heparan sulfate proteoglycan at 24 hours after the fluid load.4 Thus, before a decision regarding infusion, it is important to determine whether this patient will respond to fluid load with an adequate increase in cardiac output or stroke volume, usually by between 10% and 15% of the initial value.5 Moreover, even in the case of fluid responsiveness, increased permeability can lead to progressive tissue edema and organ dysfunction.6In patients with septic shock, fluid therapy is one of the key “golden hour” interventions. However, it can be accompanied by several potentially dangerous side effects, especially in patients with cardiac comorbidities, acute respiratory distress syndrome (ARDS), and severe “capillary leak”.11 Indeed, glycocalyx plays a key role in the physiology of microcirculation and endothelium integrity and is involved in the regulation of microcirculatory tone and vascular permeability, maintaining the oncotic gradient across the endothelial barrier, leukocyte adhesion, and migration as well as preventing the thrombosis.13 Shedding and flaking of EG result in an instant increase in free plasma concentrations of syndecan 1 (S1) and heparan sulfate proteoglycan (HSPG), which can be determined by enzyme-linked immunosorbent assay.14Nowadays, the effects of fluids on sepsis-induced degradation and shedding of endothelial glycocalyx (EG) and, thereafter, the capillary leakage resulting in the development of interstitial edema are being actively discussed.16 In sepsis, the damaged EG layer becomes thinner resulting in extravasation of proteins and fluid into the interstitial space and causing hypovolemia, hypoalbuminemia, overhydration, and tissue edema.16 Thus, critical remodeling of the endothelial system and EG triggers the mechanism of multiple organ failure. However, the relationship between EG and fluid therapy of sepsis is still a subject of debate.Sepsis and septic shock are associated with severe involvement of the endothelium and EG degradation that leads to dysregulation of homeostasis and permeability of the vascular wall causing damage to the microvasculature.The aim of our study was to assess the association of free plasma fraction of key EG components with hemodynamic and metabolic response to fluid therapy, as well as severity of pulmonary edema, in patients with septic shock.The prospective observational study was approved by the Research Ethical Committee (No. 02/06-16) of the Northern State Medical University . Written informed consent was obtained from the patient or next of kin if the patient was unconscious. Twenty-one adult patients with the diagnosis of septic shock were screened for the study entry. Three patients were excluded (2 patients due to progressing refractory shock and death during the screening period and 1 patient due to dysfunction of the thermodilution catheter), thus totally 18 patients were enrolled and underwent further analysis. The inclusion criteria were the presence of informed consent, the diagnosis of septic shock, the age of patients > 18 years, the allocation at the intensive care unit (ICU), and the requirement of mechanical ventilation. All patients were sedated and received neuromuscular blockers during fluid load test. The exclusion criteria were right heart failure, arrhythmias, and abdominal compartment syndrome. 17 The doses of vasopressors were titrated to maintain mean arterial pressure within 65-85 mm Hg.Sepsis and septic shock were diagnosed using Sepsis-3 criteria. During the study, the patients received the therapy according to the guidelines of Surviving Sepsis Campaign.2 monitor, Pulsion Medical Systems) by a triplicate 15 mL bolus of cold (<8°C) 0.9% saline solution. Cardiac index (CI), global end-diastolic volume index (GEDVI), extravascular lung water index (EVLWI), pulmonary vascular permeability index (PVPI), central venous pressure (CVP), systemic vascular resistance index (SVRI), pulse pressure variation (PPV), and stroke volume variation (SVV) were assessed using transpulmonary thermodilution and arterial pulse contour analysis.In all patients, we catheterized the internal jugular or subclavian vein with a triple-lumen central venous catheter  and the femoral artery with a thermistor-tipped arterial catheter . The arterial blood pressure was recorded from a side port of the catheter. Hemodynamic monitoring was carried out using the method of transpulmonary thermodilution  within 10 minutes. The responsiveness to fluid load was defined as an increase in cardiac output after FLT by ≥15% from the initial values, which allowed us to divide the patient data into groups of responders or nonresponders.The fluid bolus was administered within 24 hours from the onset of shock. Fluid load test (FLT) was performed by the intravenous infusion of 7 mL kgIn all patients, blood samples were taken at study baseline (before FLT), and at 2 and 24 hours after FLT. Immediately after sampling, blood was centrifuged at 2000 g for 10 minutes. The plasma fraction was immediately frozen and stored at −80°C. The components of EG including HSPG and S1 were assessed by using an enzyme-linked immunosorbent assay kit .In addition to hemodynamics and components of EG, we assessed blood gases , biochemical parameters , doses of vasopressors, Sequental Organ Failure Assessment (SOFA) scores, duration of ICU and hospital stay, and mortality at day 28. The measurements of hemodynamics, biochemistry, and blood gases were performed at baseline, immediately after FLT and at 2 and 24 hours thereafter. 2 test or Fisher exact test and expressed as patient number. For correlation analysis, we used Spearman’s rho. P values < .05 were regarded as statistically significant.For data collection and analysis, we used the Statistical Package for Social Sciences, version 17.0 software . and MedCalc software . Data distribution was assessed using the Shapiro–Wilk test. Data were presented as mean ± standard deviation (SD) or median (25th-75th percentile). The comparisons with baseline were performed using the Wilcoxon rank test. Nominal data were compared using χThe baseline clinical characteristics of study patients are presented in P < .05). Other parameters did not change significantly.The hemodynamic and metabolic variables as well as blood gases of study patients are summarized in P  = .01). Also, in the group of responders, there were increments in GEDVI at 2 hours after FLT and in EVLWI at 24 hours after FLT (P < .01). In addition, at 24 hours, the requirement of norepinephrine reduced in the responder group (P  = .01). At 2 hours after FLT, the nonresponder group demonstrated the decrease in PPV from 16% to 8% (P  = .03). At 24 hours, both mean arterial pressure and SVRI decreased whereas CI rose compared with baseline in nonresponders (P < .05). The duration of ICU and hospital stay as well as mortality did not differ significantly between responders and nonresponders.Ten patients (56%) were considered as fluid responders. We did not find significant differences in plasma fraction of S1 and HSPG, as well as in hemodynamics and clinical characteristics, between the groups of responders and non-responders at all study stages. The intragroup changes in study parameters compared to baseline in responders and nonresponders are shown in −2, n = 7) and normovolemia . At 2 hours after FLT, concentration of S1 in the normovolemic group correlated with GEDVI .Based on the GEDVI value, we identified the groups of patients with hypovolemia , whereas HSPG associated with EVLWI negatively at 24 hours after FLT . In addition, at 24 hours after FLT, the values of HSPG correlated with PPV . rho = 0.51, P  = .02). Similar association between S1 and lactate was also observed at 24 hours after the FLT .During our study, we found a relationship of EG components with hemodynamic and metabolic response to fluid therapy in septic shock. The activation of S1 and HSPG was accompanied by increased severity of noncardiogenic pulmonary edema.6 The depletion of EG at the onset of sepsis and a decrease in the concentration of its components in plasma in patients with septic shock over time have also been confirmed in a previous study by Ikeda et al.18In our study, we did not find significant changes in the concentrations of S1 and HSPG during septic shock. The possible explanation could be that at the time of inclusion into the study our patients were already diagnosed with septic shock, thus the multiorgan failure and the activation of the endothelium with glycocalyx damage has been triggered earlier.19 demonstrated that S1 is a valid marker of EG degradation, and its increment in plasma in patients with septic shock is associated with severe systemic inflammation, coagulopathy, and high mortality. Several other studies showed a significant increase in S1 in the group of patients with septic shock compared with healthy volunteers; however, the authors did not reveal a correlation between the concentration of S1 and mortality.22 In the work comparing patients with sepsis and after major abdominal interventions, the investigators found that in septic shock, the concentrations of S1 were increased significantly and rose in parallel with markers of inflammation.23 Moreover, Ostrowski et al.24 observed the relationship between the concentration of S1 and the severity of organ dysfunction in sepsis. Another component of EG, HSPG is also elevated in septic shock compared with patients after neurosurgical interventions; moreover, in patients who died within 90 days the plasma level of HSPG was 4-fold higher than in the group of survivors.22Although the increase in the plasma concentrations of S1 and HSPG after FLT in our study did not reach statistical significance, Johansson et al.Escherichia coli lipopolysaccharide (LPS), while administration of LPS was accompanied by an early decrease in the level of protein C. Recent studies have demonstrated the heterogeneity of HSPG levels depending on the pathogen strain. In particular, Gram-negative microorganisms lead to higher plasma HSPG concentrations in patients with septic shock.25However, the response of EG to infection can differ significantly. Thus, in the study of healthy volunteers who underwent endotoxemia, no significant increase in plasma S1 was obtained either after 4 hours or 6 hours from the beginning of infusion of 30 This assumption is confirmed by increased EVLWI and pulmonary vascular permeability in our study. It has been shown that too liberal infusion in septic shock causes degradation of glycocalyx and results in deterioration of its barrier function.24 In opposite, fluid restriction aiming at prevention of hypervolemia can protect EG by attenuating the release of atrial natriuretic peptide, which induces glycocalyx digestion mediated by matrix metalloproteinase.29Only 56% of patients with septic shock in our study were fluid responders, thus further fluid resuscitation was not necessary in almost half of them. In responders, we observed attenuation of tachycardia and an increase in GEDVI at 2 hours after fluid bolus. Although most effects of fluid load were transient, and there were no significant differences in studied parameters between responders and nonresponders, we found relationship between concentration of S1 and global end-diastolic volume in the normovolemic group. By contrast, fluid therapy in patients with decreased GEDVI was not accompanied by EG shedding. Indeed, in sepsis, the fluid therapy restores volume status but, if excessive, can lead to additional damage to the EG and promote capillary leak syndrome.12 During our study, the hyperdynamic state and vasoplegia were observed in nonresponders to fluid load; in parallel, they demonstrated a transient decrease in PPV. However, the value of PPV and SVV in septic shock is limited since these indexes of fluid responsiveness depend on vasopressor dose, presence of spontaneous breathing, right-heart failure, increased intra-abdominal pressure, and other factors.5 Therefore, in addition to dynamic parameters of preload, it is important to guide the fluid therapy of shock considering a complex of variables including clinical signs, lactate, arterial and venous blood gases, and volumetric monitoring.In our study, we found an association of S1 and HSPG in septic shock with hemodynamic parameters reflecting both volume status and fluid responsiveness such as GEDVI and PPV. During sepsis, several mediators including endotoxin and cytokines lead to activation of endothelial cells and their structural changes, which in turn can induce the synthesis of endothelial nitric oxide and cause vasoplegia. Degradation of heparan sulfate and hyaluronic acid also results in dilatation of the vascular bed in vivo. Thus, a positive correlation between HSPG and PPV at 24 hours after fluid load can be explained by relative hypovolemia in parallel with systemic vasodilation and endothelial damage that occurs frequently in hyperdynamic septic shock.26 This is confirmed by last studies in which authors have shown a higher plasma concentration of S1 in patients with septic ARDS, predominantly of extrapulmonary origin,15 like in the case of patients from our study.One of the main findings of our study is relationship between components of EG and severity of sepsis-induced pulmonary edema that is confirmed by positive association of S1 plasma concentration and extravascular lung water content before fluid load. Indeed, glycocalyx is a regulator of barrier integrity in the alveolar endothelium and can influence the accumulation of lung fluid.9 The possible explanation of this discrepancy can be depletion of HSPG during the clinical course and fluid therapy of septic shock. Interestingly, in our study, HSPG was associated with EVLWI negatively at 24 hours after FLT. This finding contrasts with studies where authors have demonstrated in the experimental model of sepsis-induced acute lung injury that the diffuse damage to the alveoli is related with increased plasma HSPG concentration.18 and can be explained by the effects of oxidative stress and tissue hypoperfusion on the endothelium during inflammation. In this scenario, the damage to the glycocalyx occurs, and the normal functioning of the microvasculature is disrupted, which directly affects tissue oxygenation and lactate clearance. On the other hand, according to the modern concept of shock-induced endotheliopathy (SHINE), glycocalyx discharge can serve as a protective mechanism , preventing the severity of metabolic response.10In addition, we observed that the concentration of S1 in patients with septic shock correlates with plasma lactate both before and at 24 hours after fluid load. This finding is consistent with the results of Ikeda et al.19 In addition, damage to the EG and development of pulmonary edema during excessive fluid therapy lead to a prolongation of mechanical ventilation, which increases the duration of hospital stay.It is noteworthy that during sepsis, hypoperfusion, and multiple organ failure further damage to the EG may occur. Although we have not studied the relationship of EG with clinical outcomes, several authors have shown a positive association of plasma S1 level with doses of vasopressors, the volume of fluid therapy, incidence of coagulopathy, hepatic and renal dysfunction, as well as with the duration of ICU stay and mortality.The limitations of our study include observational design, small sample size, and a relatively short period of observation with transient effects of fluid load. However, despite these limitations, our findings can be helpful for further work discovering the relationship of EG and mechanisms of cardiopulmonary dysfunction in sepsis.The initial state of EG and shedding of its components (HSPG and S1) in septic shock are interrelated with hemodynamic and metabolic disorders. After fluid load in septic shock, the accumulation of extravascular lung water during capillary leak syndrome is associated with degradation of EG components.The authors have no conflict of interest to declare.


Carbon monoxide (CO) is a vital endogenous gaseous transmitter molecule involved in the regulation of various physiological and pathological processes in living biosystems. In order to investigate the biological function of CO, many technologies have been developed to monitor the level of endogenous CO in biosystems. Among them, the fluorescence detection technology based on the fluorescent probe has the advantages of high sensitivity, excellent selectivity, simple operation, especially non-invasive damage to biological samples, and the possibility of real-time in situ detection, etc., which is considered to be one of the most effective and applicable detection techniques. Therefore, in the last few years, a lot of work has been carried out on the design, synthesis and in vivo fluorescence imaging studies of CO fluorescent probes. Furthermore, using fluorescent probes to detect the changes in CO concentrations in living cells and tissues as well as in organisms has been one of the hot research topics in recent years. However, it is still a challenge to rationally design CO fluorescent probe with excellent optical performance, structural stability, low background interference, good biocompatibility, and excellent water solubility. Therefore, this review focuses on the research progress of CO fluorescent probes in the detection mechanism and biological applications in recent years. However, this popular and leading topic has rarely been summarized comprehensively to date. Thus, the research progress of CO fluorescent probes in recent years is reviewed in terms of their design concept, detection mechanism, and their biological applications. In addition, the relationship between the structure and performance of the probes was also discussed. More significantly, we hope that more excellent optical properties fluorescent probes for gaseous transmitter molecule CO detection and imaging will overcome the current problems of high biotoxicity and limited water solubility in future. Carbon monoxide (CO) is a colorless, odorless, tasteless, and water-insoluble gaseous molecule, which binds to hemoglobin about 200 times more strongly than oxygen. When the concentration of CO in the environment is too high, it can decrease the oxygen concentration by competitively binding to hemoglobin, causing fainting and even death of living organisms ,2. ThereIn comparison to previously developed assays, such as chromogenic , electroBased on the above considerations, we systematically classified and discussed the fluorescent probes used for detecting CO over the past decade based on chemical reaction type, response mechanism, fluorophore, and biological applications . At the 2+ to Pd0, inducing the Tsuji-Trost reaction, and releasing the fluorophore. Briefly, by modulating the electron-donating ability of the hydroxyl/amine groups on the fluorophore, intramolecular charge transfer (ICT) occurs within the probe molecule, resulting in a blue/red shift in the maximum emission wavelength of the fluorescence  is a transition metal with the ability to quench the fluorescence of fluorophores through a heavy atom effect. Therefore, fluorescent probes for cyclopalladium metal complexes can be prepared by using metallic Pd coordinated to fluorophores with suitable coordination structural units . When PdThe typical ICT fluorescent probes are some fluorophores conjugated with a strong electron-giving group and an electron-absorbing group to form a robust push-pull electron system. In this reaction type, CO reduces the electron-absorbing group nitro to the electron-donating group amino, which can affect the electron density layout of the probe molecule. Thus, inducing the release of fluorophore in the system .2+ to Pd0, which further mediates the Tsuji-Trost reaction and induces changes in the fluorescence intensity. Based on the type of fluorophore, they can be divided into coumarins, naphthalimides, xanthene, near-infrared dyes, and so on.The CO fluorescent probes based on this reaction are the most rapidly developed in recent years, which mainly include allyl ether, allyl carbonate, and allyl carbamate, with the advantages of flexible construction, good selectivity, high sensitivity, and fast response time. In the design of these probes, CO was mainly reduced Pd1 based on Tsuji-Trost reaction  during cell culture and applied for living cells imaging  forms a colorless, non-fluorescent closed-loop lactone structure with a non-conjugated structure. When the analyte facilitates the deprotection of the hydroxyl group, the fluorophores are released.13, which using naphthalene fluorescein as the fluorophore and allyl carbonate as the response group, enabling the colorimetric fluorescence detection of CO in dual channels , wh, wh16 ba in 2019  34]. Mo. Mo16 baprepared  35]. No. No16 ba19 that allowed for localization to cellular mitochondria and fluorescent imaging of mice  process and a high fluorescence quantum yield  as the signal unit with good photostability  and allyl carbamate were the fluorophore and reactive group of this probe, respectively (29 and 30) to detect CO in 2018  properties for the detection and imaging of CO was firstly developed by Tang et al.  naphthalene) to detect CO in live zebrafish  vinyl complex reported a TP fluorescent probe  in 2017  53]. Th. Th39 in’s group  54]. It. It39 intive way  55]. Re. Re39 inebrafish  56]..39 in 202+ or a third substance is not required, since high concentrations of the heavy metal Pd2+ could have potential adverse effects on biological systems, such as toxicity and sensitization. For this strategy, several fluorescent probes for the detection of CO in organisms have been reported.CO is reductive and can reduce some aromatic nitro compounds in certain conditions. Hence, suitable structures of aromatic nitro compounds can be used to selectively detect CO. Compared with the methods mentioned above for detecting CO, the main strength of this strategy is that the addition of Pd43 and 44) based on CO reduction reactions  as the fluorophore and 4-nitrobenzyl as the recognition site    66]. Re. Re48 inebrafish  67]. In. In48 inCO has been demonstrated to be an essential biomarker in a variety of disease models such as inflammation, liver injury, diabetes, and cancer. Thus, the sensitive and specific monitoring of CO by fluorescence probes has irreplaceable importance for the early prediction, diagnosis and treatment of diseases. This paper reviewed the fluorescent probes for detecting CO over the last decade and outlined their chemical structures, optical properties, and bioimaging applications according to different reaction types (recognition sites). Specifically, the recognition groups of CO fluorescent probes mainly include allyl carbonate, allyl carbamate, allyl ether, nitro, etc. Furthermore, details of their emission wavelengths, detection limits, targeting capabilities and cellular tissue imaging are mentioned in the paper. All of these fluorescent probes mentioned above have a potential for the detection of CO in vivo and in vitro. More importantly, these probes have made significant advances in optical properties  and real-time monitoring CO produced in vivo.Based on the above discussion, the development of NIR, TP and ratiometric probes will be very important due to their advantages of low background interference and high tissue penetration with minimal damage. In addition, the sensing properties of organelle-targeted fluorescent probes remains to be enhanced, including the sensitivity and selectivity, which are critical for analyzing endogenous CO in vivo and providing insight into the physiological and pathological processes related to human diseases. The above-mentioned photoacoustic CO probes also exhibit great potential, because they can produce thermoelastic expansion with amazing tissue penetrating ability and are non-invasive during imaging, thus avoiding complex invasive surgical operations. Furthermore, fiber optic probes have significant advantages such as small size, insulation, fast response time, immunity to electromagnetic interference, high measurement accuracy, and good bioaffinity, which make them have important applications in biomedicine ,69,70. IHowever, the design of CO fluorescent probes needs to take into account their biocompatibility, water solubility, resistance to other reactive oxygen species (ROS) interferences and poor stability drawbacks. Thus, in future CO probe designs, these problems can be effectively addressed by grafting natural materials to construct nanosensors or copolymerizing them into polymer probes. These methods are believed to enhance their stability and biosafety, at the same time, they will help to solve many defects of small molecular fluorescent probes.We have always thought that the exploration of fluorescent probes for detecting CO, particularly in biosystems, will be one of the significant research directions to further elucidate the vital function of ROS in various biological processes. We hope that this review will draw deeper attention to CO and provide empirical references for the design and synthesis of subsequent CO fluorescent probes.


Adropin plays an important role in the regulation of energy homeostasis and metabolism. It enhances glucose tolerance, improves insulin resistance, ameliorates lipid metabolism, and has antihyperlipidemic activity. It has previously been detected in various organs, including the pancreas. Our aim was to investigate whether adropin affects pancreas exocrine secretion. The influence of different doses of adropin was tested in basal and stimulated conditions . In addition, the effect of adropin has been studied under vagotomy and capsaicin deafferentation conditions. Our results indicate that adropin inhibits pancreas exocrine secretion both in the basal and stimulated conditions, whereas vagotomy and deafferentation abolish the pancreas responses to adropin. Based on the obtained results, it can be assumed that the effect of adropin on the pancreas is related to an indirect vagal mechanism. Nevertheless, this hypothesis requires further verification.p < 0.001 for adropin group and p < 0.01 for CCK-8 and vagal stimulation groups), and the protein outputs were reduced by the administration of ADR 10 µg/kg BW  and 20 µg/kg BW . The 10 µg/kg BW dose of ADR reduced the trypsin output in the CCK-8 group (p < 0.01), and the highest ADR dose reduced the trypsin output in the CCK-8 (p < 0.001) and vagal stimulation (p < 0.01) groups. In conclusion, adropin in the analyzed doses exhibits the negative feedback pathway. This mechanism seems to participate in the regulation of pancreatic juice secretion via an indirect vagal mechanism.The aim was to investigate the potential effect of adropin (ADR) on pancreatic–biliary juice (PBJ) secretion  in a rat model. The animals were divided into control and five experimental groups: adropin, CCK-8 (CCK-8 stimulation), capsaicin (capsaicin deactivation of afferents), vagotomy (vagotomy procedure), and vagal stimulation . The experiment consisted of four phases, during which vehicle (0.9% NaCl) and three ADR boluses  were administered i.v. every 30 min. PBJ samples were collected from each rat at 15 min intervals after boluses. Exogenous ADR failed to affect the pancreatic responses after vagotomy and the capsaicin pretreatment and reduced the PBJ volume, protein outputs, and trypsin activity in the adropin, CCK-8, and vagal stimulation groups in a dose-dependent manner. In all these groups, volume of PBJ was reduced only by the highest dose of ADR ( ENHO) expressed mostly in the brain and liver [In 2008, Kumar et al.  describend liver . ADR34-7nd liver ,6.ADR plays an important role in the regulation of energy homeostasis and metabolism. It enhances glucose tolerance, improves insulin resistance, and promotes the utilization of carbohydrates over fat in muscles ,8. ExogeThere is little information in the available databases about the relationship between adropin and gastrointestinal tract function. It is limited to a few reports. The gastroprotective potential of ADR was demonstrated in rats with indomethacin-induced gastric ulcers . On the ENHO mRNA expression in rat pancreatic islets and very low expression in INS-1E cells [The presence of ADR in the digestive tract was detected in the duodenum and jejunum. It was located in crypts, enterocytes, submucosa, muscularis externa, Brunner’s gland, and myenteric and submucosal plexus nerve . Aydin e1E cells . These d1E cells .Taking into account the very limited information on the role of adropin in the physiology of the gastrointestinal tract, the aim of our research was to investigate the potential effect of ADR on pancreatic juice secretion in a rat model in different conditions.All experimental procedures performed in this study were approved by the Local Ethical Committee for Animal Experiments .n = 6 animals:Control (four 0.9% NaCl boluses);Adropin (adropin boluses);CCK-8 stimulation (CCK-8 stimulation and adropin boluses);Capsaicin (capsaicin pretreatment and adropin boluses);Vagotomy (vagotomy and adropin boluses);Vagal Stimulation .In vivo studies were performed on 36 male Wistar rats weighing 200–220 g. The rats were housed in cages in standard conditions at a controlled room temperature of 22 °C (±10%) and humidity of 55% (±10%) with a normal circadian rhythm: a 12 h day/night cycle. During the one-week period of acclimation to the new environment, food  and water were available ad libitum. After the acclimatization period, the rats were randomly divided into six groups, one control and five experimental, each containing The night before the in vivo experiment, the animals were fasted, but water was not limited. All experiments were carried out at the same time in the morning. Surgical operations were performed under general anesthesia with an intramuscular (i.m.) injection of ketamine + xylazine . For intravenous (i.v.) infusion, the right external jugular vein was prepared, and inserted tubes (silicone) were secured with ligatures. Continuous i.v. infusion of 0.9% NaCl  started immediately after cannulation and was maintained until the end of the experiment, except the i.v. application of adropin boluses. The bile and pancreatic juice mixture (PBJ) was collected from tubing (polyethylene) inserted in the common pancreatic–biliary duct after midline laparotomy. For the reintroduction of PBJ, second tubing was inserted into the duodenum through the pylorus. The anesthesia was maintained during the experiment by intraperitoneal administration of anesthetics, and rats’ body temperature was continuously monitored and maintained by heating lamps. The involvement of vagal nerves was examined following subdiaphragmatic vagotomy and capsaicin deafferentation, as described previously ,21. The Collection of PBJ started immediately after the surgery. PBJ was collected at 15 min intervals into 1.5 mL polyethylene tubes kept on ice. In four phases of the experiment vehicle  and three adropin boluses  were administered i.v. every 30 min . The plaCollected PBJ samples were used for further analysis. The volume of the PBJ samples was measured, and PBJ was aliquoted into 0.1 mL samples and stored in polypropylene tubes at −20 °C. The total protein content was estimated with the Lowry method in 96-well microwell plates according to the methods described previously [p < 0.05 were considered significant.The results are expressed as means ± SD. The statistical analysis was conducted using the statistical package GraphPad Prism 5.00 . Statistical significance for all groups was investigated via one-way repeated-measures ANOVA, indicating significant differences. Mean differences between Adropin 0  and the others were analyzed by Dunnett’s post-hoc test. Differences with p ˂ 0.01) (p ˂ 0.001) and in the vagus-nerve-stimulated animals . No significant changes in the PBJ volume were observed after adropin administration in rats after vagotomy and capsaicin deactivation of afferents  A. Signiffferents A.The protein outputs were reduced by the administration of 0, 5, 10, and 20 µg/kg BW ADR. The protein outputs were 0.64, 0.59, 0.44, and 0.39 mg/100g BW/15 min, respectively, in the basal conditions, 0.76, 0.69, 0.60, and 0.55 in the CCK stimulation group, and 0.69, 0.61, 0.58, and 0.56 in the vagus-nerve-stimulated animals. No responses to the application of adropin were observed in rats after vagotomy and deafferentation with capsaicin. The protein outputs at the doses of 0, 5, 10, and 20 µg/kg BW ADR were 0.37, 0.37, 0.38, 0.40 mg/100 g BW/15 min, respectively, in the vagotomized rats and 0.48, 0.46, 0.46, and 0.45 in the rats subjected to the capsaicin pretreatment B.The effect of ADR on the trypsin output was dose related. The highest dose of ADR reduced the trypsin output significantly in CCK (0.98 vs. 0.67) and vagus-nerve-stimulated (0.74 vs. 0.59) rats. No statistical differences in trypsin outputs were observed after the vagotomy and deafferentation with capsaicin. The trypsin outputs at the ADR doses of 0, 5, 10, and 20 µg kg/BW were 0.49, 0.48, 0.48, 0.47 U/100g BW/15 min, respectively, in the vagotomized rats and 0.41, 0.39, 0.39, and 0.38 in the capsaicin pretreatment group C.The gastrointestinal tract (GI tract) and pancreas are known to provide hormonal signals to the central nervous system. These hormones include ghrelin, obestatin, gastric leptin, apelin, and nesfatin-1 secreted by the stomach and intestines and released in small amounts from the endocrine pancreas ,27,28,29ADR, involved in regulating the body’s energy homeostasis, is secreted by many types of tissues in the human body . First, Currently, there is no information in the scientific literature about the role of adropin in pancreatic exocrine functions. Our study shows for the first time that the administration of adropin influences pancreatic juice secretion, as we demonstrate that exogenous adropin can reduce the volume of pancreatic juice secretion and decrease protein outputs and trypsin activity in a dose-dependent manner. Adropin inhibited the exocrine function of the pancreas in both the basal and CCK-8-stimulated conditions. The strongest effect of adropin administration on the PBJ volume was observed after the application of the highest dose of ADR (20 µg/kg BW), while the most potent effect on the protein and trypsin outputs was noted after the administration of 10 and 20 µg/kg BW.In the last two decades, our team studied the influence of newly discovered hormones on pancreatic juice secretion. In earlier studies, we found that the i.v. administration of exogenous ghrelin and apelin decreased ,32, whilMoreover, our previous studies showed that the i.v. administration of apelin-13 boluses decreased pancreatic blood flow . In turnThe vagus nerve participates in the regulation of pancreatic juice secretion. Several studies have shown that electrical vagal stimulations induced the release of CCK-8 into the circulation ,23,46. OIn conclusion, adropin administered in the analyzed doses exhibits a negative feedback pathway. This mechanism seems to participate in the regulation of pancreatic juice secretion via an indirect vagal mechanism. However, the current knowledge about adropin with its key role and mechanism in the regulation of many physiological and pathophysiological conditions, including pancreatic function, is insufficient, and this issue should be further investigated not only in the rat model but also in other non-human-model animals.