Background and objectives: Research investigating hydration strategies specialized for women’s soccer players is limited, despite the growth in the sport. The purpose of this study was to determine the effects of fluid balance and electrolyte losses in collegiate women’s soccer players. Materials and Methods: Eighteen NCAA Division I women’s soccer players were recruited , including: 3 forwards (FW), 7 mid-fielders (MD), 5 defenders (DF), and 3 goalkeepers (GK). Players practiced outdoor during spring off-season training camp for a total 14 practices (WBGT: 18.3 ± 3.1 °C). The main outcome measures included body mass change (BMC), sweat rate, urine and sweat electrolyte concentrations, and fluid intake. Results: Results were analyzed for comparison between low  and moderate risk environments for hyperthermia  as well as by field position. The majority (54%) of players were in a hypohydrated state prior to practice. Overall, 26.7% of players had a %BMC greater than 0%, 71.4% of players had a %BMC less than −2%, and 1.9% of players had a %BMC greater than −2% . Mean %BMC and sweat rate in all environmental conditions were −0.4 ± 0.4 kg (−0.5 ± 0.6% body mass) and 1.03 ± 0.21 mg·cm−2·min−1, respectively. In the MOD environment, players exhibited a greater sweat rate (1.07 ± 0.22 mg·cm−2·min−1) compared to LOW . By position, DF had a greater total fluid intake and a lower %BMC compared to FW, MD, and GK . FW had a greater sweat sodium (Na+) (51.4 ± 9.8 mmol·L−1), whereas GK had the lowest sweat sodium (Na+) (30.9 ± 3.9 mmol·L−1). Conclusions: Hydration strategies should target pre-practice to ensure players are adequately hydrated. Environments deemed to be of moderate risk of hyperthermia significantly elevated the sweat rate but did not influence fluid intake and hydration status compared to low-risk environments. Given the differences in fluid balance and sweat responses, recommendations should be issued relative to soccer position. Competitive soccer players move continuously during match play, covering large distances 8–12 km) using both aerobic and anaerobic energy systems 2 km usin,4. FédérCurrent hydration guidelines exist to protect players from illness and injury as a result of hypohydration. Hydration requirements vary across each sport, as well as the position within each sport due to differences in exercise mode and intensity, and environmental conditions . ResearcA recent publication by Belval et al. (2019) suggested that personalized hydration strategies are necessary to promote adequate hydration and optimize performance based on the exercise intensity, fluid availability, and environmental condition of each sport . In compTo our knowledge, no study has examined the position-related differences in overall fluid balance in women’s soccer to assess individualized fluid and electrolyte needs. The purpose of this study was to describe the fluid balance, sweat rate, and electrolyte losses in a single NCAA Division I women’s soccer team. We hypothesized that fluid and electrolyte needs would be dependent on field position with forward and mid-fielders requiring additional fluid intake due to their physical activity pattern. In addition, we examined the effect that environmental conditions had on the relationship between fluid balance and field position. Eighteen players were recruited from a single NCAA Division I women’s soccer team . Players were grouped according to field position as follows: 3 forwards (FW), 7 mid-fielders (MD), 5 defenders (DF), and 3 goalkeepers (GK). Players were free of any chronic condition and injury and were cleared by their team physician to participate in play. The study was approved by the University Institutional Review Board and all players signed a written informed consent prior to any assessment .The study took place over 7 weeks of practice during a spring off-season training camp (March to mid-April). Players were on-field training every Tuesday and Thursday morning (0730–0900) and Friday afternoon (1530–1700). Due to the accessibility of players and the training schedule, data for this study were only collected on 2 of the 3 practices per week, for a total of 14 evaluated practices. Of the two practices observed, a morning practice (Tuesday and Thursday) and evening practice (Friday) was included. WBGT and relative humidity (RH) varied between 10.2–23.6 °C and 29–86%, respectively. Environmental stress was categorized as a low and moderate risk of hyperthermia using the combination of WBGT and RH . Accordiad libitum and consisted of either water and/or carbohydrate-electrolyte solution (CES). Practice assessments involved environmental conditions and physiological monitoring including changes in body mass, fluid intake, urine and sweat assessments, and heart rate (HR). Environmental conditions (WBGT and RH) were measured before and after practice . Practice consisted of a 10- to 15-min warm-up and 4–5 sets of 15 min scrimmages with a 1- to 2-min break between scrimmages. Study investigators had no influence on the training or sport-specific performance testing. Medical and athletic training staff were present throughout each practice. During the rest period, fluids were provided by the athletic trainers Body mass was assessed using a scale with a precision of 0.1 kg  before and after each practice. Hydration status was represented as %BMC, and fluid balance was calculated as the change in body mass (after toweling dry and urinating), accounting for fluid intake and urine output. In addition, hydration status was categorized by %BMC as euhydrated , mild hypohydration , and moderate hypohydration  to further detect the positional differences , despitead libitum during the practice rest periods. The beverages provided were water (WATER) and/or carbohydrate-electrolyte solution (CES) according to the player’s individual preference during practice. The CES contains 150 mg sodium and 21 g of carbohydrate per serving . Fluid bottles, WATER and CES, were individually labeled with players’ names to ensure an accurate estimate of fluid intake for each player and beverage ingested. Each position and player had equal access to both WATER and CES and the athletic training staff were instructed not to influence beverage selection. Overall fluid intake was determined by pre- and post-practice water bottle weights using food preparation scales . Players were encouraged to ingest all fluids that entered the mouth so that accurate measures of total fluid intake could be determined. Players self-selected beverages to drink Players were instructed to completely empty their bladder and provided a urine sample prior to body mass measurements immediately before and after practice. Urine specific gravity (USG) was measured using a spectral refractometer . For the purpose of this manuscript, a USG of ≥1.020 will be considered to be an impaired hydration status as suggested by Casa et al. (2000) . Urine w−2·min−1). Sweat was analyzed for electrolyte concentrations (sodium (Na+), potassium (K+), and chloride (Cl-)) similarly to urine. Regional sweat electrolyte concentrations and sweat rate were corrected to represent whole body losses according to Baker el al. (2019) [Sweat was collected on the lower back using the technical absorbent patch technique . The ski. (2019) ,24.n = 3), MD (n = 5), and DF (n = 3). Average heart rate (HRavg) and maximal heart rate (HRmax) were determined for the time during practice. Average exercise intensity was calculated as the HRavg divided by HRmax, multiplied by 100 (%HRmax). HRmax was determined by the maximum HR across 14 days of practice for each player.Heart rate was monitored continuously using a Bioharness BH3  worn around the chest during each practice. Eleven of the 18 players wore a Bioharness as designated by the head coach and included FW (2) was used to analyze BMC categories  by position. Pearson product moment correlations (r) were used to examine the relationships between outcome variables. The total sample size for sweat and urine assessments is displayed in p < 0.05.Data were analyzed using JMPro 14 . Outcome variables included %BMC, fluid intake, sweat and urine electrolytes, USG, sweat rate, and HR parameters. One- and two-way analysis of variance (ANOVA) were used to analyze outcome variables across field positions  and environmental conditions (LOW and MOD) with Student-t post-hoc tests to examine group differences. Chi-square (Xp = 0.5). Post-exercise urine sodium concentration (111.3 ± 57.4 mmol·L−1) was significantly lower compared to pre-exercise urine sodium concentration . Mean %BMC and total fluid intake were −0.5 ± 0.5% and 630 ± 312 g, respectively. Mean sweat rate was 1.03 ± 0.21 mg·cm−2·min−1 and sweat sodium concentration was 44.5 ± 10.4 mmol·L−1. chi2 = 21.33, p = 0.002).Categorial hydration status data by position across all practices is displayed in p < 0.001). In addition, DF had the greatest total fluid intake , which was characterized by the greatest CES  and the least WATER  ingestion compared to other positions. In addition, total fluid intake was significantly associated with %BMC across positions . GK had significantly lower sweat (Na+) compared to FW, MD, and DF . FW had significantly greater sweat (Na+) compared to MD and greater sweat (Cl-) compared to MD and DF (p < 0.001). Sweat (K+) was greatest in DF compared to FW and MD (p = 0.03). However, sweat rate was similar across all practices and all positions . MD had significantly greater HRmax (216 ± 18 bpm) compared to FW (207 ± 10 bpm) and DF . In addition, FW had a greater average exercise intensity (74.5 ± 8.2% HRmax) compared to MD (67.6 ± 5.8% HRmax) and DF . Lastly, exercise intensity (%HRmax) was significantly associated with sweat (Na+)  across all positions.An ancillary subset of 11 players wore heart rate monitors during the 14 practices . HRavg was similar between positions . In addition, these outcomes exhibited similar results across all positions in LOW and MOD environments (−2·min−1) resulted in a significantly greater sweat rate compared to LOW environment  across all players  began practice in a hypohydrated state. During practice, 71.4% of players had a %BMC < −2%; 26.7% of players had no body mass deficit; and 1.9% of players had %BMC ≥ −2%  demonstrating the effectiveness of the players to adequately hydrate during practice. Environments that are considered of a moderate hyperthermia risk significantly elevated sweat rates compared to low risk environments; however, environmental conditions had no influence on fluid intake and hydration status across positions. By position, we observed that DF had a significantly greater total fluid intake and a lower %BMC compared to other positions. FW exhibited greater sweat sodium and chloride concentrations; whereas GK had the lowest sweat electrolyte concentrations compared to other positions. Therefore, positional differences existed in fluid balance and sweat responses, suggesting variable hydration requirements across player positions to maintain both fluid and sodium balance. Players seem to understand the importance of fluid balance during training but may underestimate the importance of pre-practice fluid intake on overall training hydration status. Previous research suggests that USG is a good indicator of pre-practice hydration status, specifically, a USG of >1.020 can be considered chronically hypohydrated . In the The availability of fluids has been suggested to be a key element associated with total fluid intake in team sports . KildingExercise characteristics and running distance vary by position, which are potential factors that affect fluid balance in soccer . In genep = 0.004) between sweat sodium concentration and exercise intensity, suggesting that exercise intensity is a factor associated not only with sweat rate , but overall sweat electrolyte losses. Given the evidence of the difference in fluid balance outcomes by on-field position, recommendations for fluid intake during training and competition in women’s soccer should be based on on-field exertion characteristics. In this study, the sweat electrolyte concentrations are considered normal in women’s soccer players. Under optimal conditions, an athlete’s sweat rate and electrolyte composition would be known, and with dietary fluid and electrolyte intake, athletic training staff could provide supplemental sodium containing foods or beverages if sodium balance is not achieved. Under normal conditions, athletic trainers and nutritionists should encourage normal dietary intake of fluid and electrolytes; however, supplemental fluid and sodium intake may be encouraged in athletes with high sweat and/or sodium losses, or when athletes have longer practice or practice is conducted under higher thermal stress. Future research could determine intensity thresholds, in combination with environmental conditions, to better estimate fluid intake recommendations across all positions.Greater sweat sodium and chloride concentrations were observed in FW, whereas the lowest concentrations were found in GK. One possible explanation for this might be that GK had a large proportion of water intake, possibly lowering serum and sweat osmolality, which results in a lower sweat sodium concentration. In addition, sweat sodium concentrations have been shown to be elevated at greater exercise intensities . We examEstimated sweat rate was similar between positions across all practices. Previous studies have reported that sweat rate cannot be easily explained by fluid intake, pre-exercise hydration status, or variations in body mass within players ,36. Our A regional sweat patch technique was used to determine regional sweat rate and electrolyte concentrations, which have been shown to represent whole-body sweat electrolyte losses . Mean swAlthough this study has many strengths including on-field, real-world implications for hydration across diverse environmental conditions, accurate measures of fluid intake by bottle weight changes, repeated measures for each player, and continual assessments of heart rate, and exercise intensity, there are marginal limitations that must be mentioned. First, this study had a small sample size for field-based research, particularly when analyzed by positions. Moreover, although we measured exercise intensity, running distance during training was not recorded, forcing us to make several inferences from other research to describe the players according to position. However, as mentioned above, we did continually assess heart rate responses for a large subset of the players, allowing us to express our results by intensity and time of practice, which should better represent overall effort compared to describing distance alone. In addition, it might not be appropriate to categorize participant’s hydration status after practice as euhydrated, mildly hypohydrated and moderately hypohydrated based on the body mass change because we did not ensure an adequate starting hydration level by forcing fluid intake prior to practice similar to laboratory-based research. This is especially true considering more than half of players started practice in a hypohydrated state according to USG (>1.020). Given this limitation, we applied relative hydration categories to show the magnitude of body mass change to further demonstrate positional differences. A further limitation to this study is the way in which fluid intake was tracked: 2 labeled bottles, 1 WATER and 1 CES, for each player. Normally, each player has a single bottle with their names and beverage of choice. By offering two bottles (CES and WATER) per player at every practice, we may have inadvertently altered drinking behavior . AdditioIn the present study, 54% of players were hypohydrated prior to the practice and the majority of players maintained adequate fluid balance during practice under variable environmental stress. We found the environment to be most likely to influence sweat rate, but not fluid intake and hydration status. By position, DF exhibited a greater total fluid intake, resulting in lower fluid balance deficits. MD appeared to be at the greatest risk of hypohydration with more occurrences of %BMC over −2% and a higher maximal heart rate. FW had the greatest sweat sodium concentrations, whereas, GK had the lowest, suggesting additional sodium should be ingested in more intensive positions. This is the first study to describe the positional differences in fluid balance of collegiate women’s soccer players. These observations in positional fluid balance differences suggest that positional exercise intensity and fluid and electrolyte needs should be considered in future hydration recommendations. In addition, hydration strategies should emphasize pre-practice hydration to ensure that players are prepared for the demands of the sport.


Rodent models have contributed significantly to the understanding of haematological malignancies. One important model system in this context are patient-derived xenografts (PDX). In the current study, we examined 20 acute leukaemia PDX models for growth behaviour, infiltration in haemopoietic organs and sensitivity towards cytarabine. PDX were injected intratibially (i.t.), intrasplenicaly (i.s.) or subcutaneously (s.c.) into immune compromised mice. For 18/20 models the engraftment capacity was independent of the implantation site. Two models could exclusively be propagated in one or two specific settings. The implantation site did influence tumour growth kinetics as median overall survival differed within one model depending on the injection route. The infiltration pattern was similar in i.t. and i.s. models. In contrast to the s.c. implantation, only one model displayed circulating leukaemic cells outside of the locally growing tumour mass. Cytarabine was active in all four tested models. Nevertheless, the degree of sensitivity was specific for an individual model and implantation site. In summary, all three application routes turned out to be feasible for the propagation of PDX. Nevertheless, the distinct differences between the settings highlight the need for well characterized platforms to ensure the meaningful interpretation of data generated using those powerful tools. Tumour inhibition on a particular day (T/Cx) was calculated from the median RTV of a test group and the median RTV of a control group multiplied by 100, as shown by the following equation: T/Cx [%] = median RTVx treated group/median RTVx control group*100. The minimum T/Cx [%] value recorded for a particular group during an experiment represented the maximum anti-tumour activity for the respective compound and were thereby defined as optimal T/C. All experiments were performed according to the relevant animal welfare guidelines published by FELASA and GV-SOLAS in an AAALAC accredited animal facility. Tumour volume and body weight were determined twice per week. Percentage of human CD45+ cells in PB once a week. The calculation of the optimal T/C value was performed as described above for tumour volume. Mice were treated with cytarabine  or vehicle (PBS) for five consecutive days. Treatment commenced upon disease onset: for i.t. implanted animals this was determined as > 2% human CD45+ cells in PB. For s.c. implanted animals the starting median absolute tumour volume was 100 mm³. Beside those read-outs OS served as a read-out for both implantation techniques. The relative volume of an individual tumour on day X (RTVx) was calculated by dividing the absolute volume  in an AAALAC accredited animal facility. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of the regional council . The analyses were performed according to the guidelines of the Declaration of Helsinki and good clinical practice. All patients gave their written informed consent for institutional-initiated research studies and analyses of clinical outcome studies conforming to our institutional review board guidelines. All experiments were approved by the ethical commission of the Albert Ludwig University Freiburg . t test, two-tailed and one-way ANOVA followed by Tukey’s multiple comparisons test were used to calculate all reported p-values. Log-rank [Mantel–Cox]-test was used to calculate overall survival. Descriptive analyses were assessed whenever appropriate and were obtained using GraphPrism software [Student’s software  The mutational data of the PDX models were retrieved via the online compendium from Charles River  and compHeat maps and PCA plots were generated using the ClustVis web tool . CenteriWe took advantage of our well characterized leukaemia PDX panel and evaluated the influence of the injection site on different parameters of the PDX growth behaviour. Taken together, the features of an individual PDX model are dictated by the genetic make-up of the model itself and the tumour microenvironment modified in the current study via the engraftment site. Finally, the question of which in vivo model will most precisely reflect the patient situation ultimately may depend on the focus of the scientific question.


Here, we describe a combination strategy of black phosphorus (BP)-based photothermal therapy together with anti-CD47 antibody (aCD47)-based immunotherapy to synergistically enhance cancer treatment. Tumour resistance to immune checkpoint blockades in most cancers due to immune escape from host surveillance, along with the initiation of metastasis through immunosuppressive cells in the tumour microenvironment, remains a significant challenge for cancer immunotherapy. aCD47, an agent for CD47/SIRPα axis blockade, induces modest phagocytic activity and a low response rate for monotherapy, resulting in failures in clinical trials. We showed that BP-mediated ablation of tumours through photothermal effects could serve as an effective strategy for specific immunological stimulation, improving the inherently poor immunogenicity of tumours, which is particularly useful for enhancing cancer immunotherapy. BP in combination with aCD47 blockade activates both innate and adaptive immunities and promotes local and systemic anticancer immune responses, thus offering a synergistically enhanced effect in suppression of tumour progression and in inducing abscopal effects for inhibition of metastatic cancers. Our combination strategy provides a promising platform in which photothermal agents could help to enhance the therapeutic efficacy of immunotherapy. By combining photothermal therapy and immunotherapy, scientists have improved cancer treatments. In cancer immunotherapy, the body’s immune system is artificially boosted to fight cancer. Some cancers, however, can protect themselves from immunotherapies by stimulating immune checkpoint targets, which regulate the immune system and prevent it from attacking non-cancerous cells. Checkpoint blockade immunotherapy helps the immune system to recognise and attack cancer cells. However, it is ineffective in treating patients suffering from most solid tumour cancers like melanomas. Now, a team of scientists, led by Paras Prasad from the State University of New York, have used a combination of black phosphorus-based photothermal therapy and antibody immunotherapy to enhance the therapeutic efficiency of the treatment. By exerting a synergistically enhanced antitumor effect, the technique has proved effective in suppressing the proliferation and growth of cancer cells. Currently, FDA-approved immune checkpoint blockades target the molecules PD-13, PD-L15 and CTLA47, which play pivotal roles in the regulation of T-cell activities, for the treatment of solid, metastatic and haematological tumours. However, the therapeutic efficiency of immune checkpoint blockades remains relatively low for most cancer patients, especially those suffering from solid tumours.Cancer immunotherapy, also called immuno-oncology, is a treatment strategy that artificially boosts the body’s own immunity to battle cancer. Immune checkpoint blockade is the most promising therapy for the activation of antitumor immunity that targets the immune checkpoint, which is the key regulator overexpressed in cancer cells to escape the body’s immune system9. TAMs, as one of the most abundant immune cells, are dependent on the factors present in the tumour microenvironment10 and characterised by their protumoural functions, such as tumour growth promotion, progression of invasion and metastasis and even induction of angiogenesis11. TAMs can secrete a series of chemokines and cytokines that directly or indirectly suppress the activity of CD8+ and CD4+ T cells to facilitate tumour immune escape12. Macrophages infiltrating the tumour microenvironment are composed of two subtypes: M1-like and M2-like macrophages. The former are classically activated macrophages that exert antitumour functions, such as antigen presentation and lysis of cancer cells. The latter are alternatively activated macrophages, exhibiting protumoural effects by directly participating in cancer cell proliferation, invasiveness, and progression along with immunosuppression15. The repolarization of TAMs from M2-like to M1-like macrophages has been shown to be a promising target for controlling tumour progression16.A lack of recognisable antigens on the tumour cell surface, defects in tumour-specific antigen presentation, the absence of infiltration of cytotoxic T lymphocytes (CTL), severe T-cell exhaustion and immune escape from host surveillance contribute to the initial resistance to immune checkpoint blockades. The immunosuppression status within most tumours is a major challenge because no efficient and durable antitumour immune response can be established or maintained. Even worse, these immunosuppressive cells can promote tumour immune escape, progression and metastasis. Extensive evidence has demonstrated that the major immunosuppressive cells promoting tumour progression comprise myeloid-derived suppressor cells, tumour-associated macrophages (TAMs), regulatory T cells and T helper 17 cells17. By interacting with signal regulatory protein-alpha (SIRPα)18, which is expressed on macrophages, CD47 can realise the function of ‘don’t eat me’. Binding of CD47 of tumour binding to SIRPα inhibits the phagocytosis of macrophages to evade innate immunological eradication as well as subsequent adaptive immunity20. CD47/SIRPα axis blockade agents such as anti-CD47 antibodies (aCD47) have demonstrated excellent efficacy against lymphocytic leukaemia17, hepato cellular carcinoma21, acute myeloid leukaemia19, aggressive metastatic leiomyosarcoma22 and solid tumours23. Currently, a CD47 antagonist (Hu5F9-G4) has completed its phase 1 trial for treating patients with haematological malignancies  and solid tumours (NCT02216409). However, the blockade of the SIRPα-CD47 interaction cannot induce sufficient phagocytic activity and antitumor immune response24, resulting in failure of the clinical trial (NCT02641002). To improve the response rate of aCD47, researchers have conducted several clinical trials to evaluate the efficacy of aCD47 combined with other anticancer drugs, such as aCD47 with rituximab in treating lymphoma (NTC02953509), azacitidine in treating acute myeloid leukaemia or myelodysplastic syndrome (NTC03248479) and cetuximab in treating advanced colorectal cancer (NTC02953782). To enhance the therapeutic effect of the aCD47 antibody, researchers have employed appropriate combination strategies with additional stimuli, e.g., chemotherapy and phototherapy, to potentiate antitumour immunity26. Thermal tumour ablation, as a minimally invasive therapy, is one of the most commonly used treatments for a variety of solid tumours, such as hepatic carcinoma, renal carcinoma and osteoma. Hyperthermic ablation is also an alternative technique for patients with failure of chemotherapy or radiotherapy and people who are not good candidates for surgery27. In photothermal therapy (PTT), an external light source  with strong tissue penetration is absorbed by photothermal nanomaterials for conversion to thermal energy, resulting in cell death by necrosis through increased local temperature28. Increasing studies have reported that PTT causes immunogenic cell death, releasing damage-associated molecular patterns30 that act as endogenous danger signals31, hence improving tumour immunogenicity32.Macrophages as effector cells of cancer immunotherapy may have therapeutic potential because of their potent phagocytosis ability. CD47, a self-recognition protein, is ubiquitously expressed on human cells and upregulated in many different tumour cells49. Ultrathin 2D black phosphorus (BP) nanosheets (NSs), as a metal-free photothermal agent that shows a high efficiency for converting NIR to heat, have attracted increased attention in biomedical applications because they are easily biodegradable and biocompatible52. BP NSs are not stable and are gradually degraded into nontoxic small molecular weight substances such as phosphates and phosphonates53. Phosphorus in the form of phosphate participates in almost all cellular processes54. For instance, the sugar-phosphate backbone is the main component of the structural framework of DNA and RNA. Living cells need phosphate for transferring cellular energy with ATP. An adult human contains on average ~700 g of phosphorus55. Given the excellent biocompatibility properties of BP, it is possible that these ultrathin 2D inorganic NSs have broad potential in the field of biomedical applications as well as in clinical translation.Results on graphene have inspired increased research interest in two-dimensional (2D) materialsHere, we found that BP-based hyperthermic ablation could not only directly destroy tumour cells, releasing tumour-associated antigens and alarmin, but could also act as an effective specific immunological stimulator and boost the poor immune-stimulating properties of tumour antigens, relieving immunosuppression in a tumour microenvironment. BP in combination with immune checkpoint blockade via aCD47 effectively obstructs tumour progression, exerting a significant synergistic effect. We further found that BP plus aCD47 could induce repolarization of TAMs to M1-like macrophages, which not only blocks the CD47/SIRPα interaction, promoting phagocytosis, but also potentiates the local cross-presentation of tumour-associated antigens, thus activating tumour-specific T-cell activity and further inducing the abscopal effect for fighting metastatic cancer , and Raman techniques. The HRTEM image showed clear lattice fringes, with an interatomic distance of 0.22 nm  and transmission electron microscopy (TEM) were used to characterise the morphology of BP NSs. The lateral size of the BPNSs was shown to be 100–200 nm by the TEM image Fig. . The thi nm Fig. , which cern Fig.  and SAEDern Fig.  further ern Fig.  consistece Figs.  and S2.FBased on the analyses of the morphological, chemical and crystal structure, we can conclude that the exfoliated BPNSs maintain the corresponding chemical and crystalline state of BP.−1 cm−1 at 808 nm based on the Beer–Lambert law . The temperature gradually increased to a saturation level as the irradiation time increased  under laser irradiation, the temperature was maintained at ~30 °C, which was insufficient to induce any tissue damage57.Considering the potential ability of photothermal agents to increase tumour immunogenicity, we evaluated whether PTT with BP can trigger a strong immune response to recognise tumour-specific antigens. We first tested the immunological effects of photothermal ablation in BALB/c mice bearing subcutaneous B-cell lymphoma A20 tumours subcutaneously. When the tumour volume reached ~100 mmmia Fig. 57. When +CD11b+), the direct precursors of macrophages and dendritic cells, within the tumours was observed compared to that in the control group, suggesting that more monocytes may be recruited to the ablated tumour tissues for phagocytosis and presentation of tumour-specific antigens to initiate and regulate immune responses , which play a key role in promoting the apoptotic death of cancer cells59, infiltrated the tumour after BP treatment with NIR irradiation than that in the control group  model to evaluate the antitumour effect of aCD47 or BP plus aCD47 in immunotherapy research. The A20 cell lines are B-cell lymphoma lines and have histological features of lymphoma. Subcutaneous implantation of A20 requires a large number of cells to form a subcutaneous tumour after 10 days. In addition, subcutaneous inoculation facilitates intratumoural injection and measurement of tumours. Encouraged by the excellent immunological effect promoted by BP hyperthermic ablation, we thus hypothesised that such a specific immunity stimulator may produce a synergistic enhancement effect with the aCD47 checkpoint inhibitor. Next, to test the synergistic antitumour efficacy of BP-based PTT with aCD47, we subcutaneously inoculated 5 × 106 B-cell lymphoma cells (A20) in the right flank of Balb/c mice. Ten days later, when the tumour volume reached ~100 mm3, the Balb/c mice were randomly assigned to five groups: untreated (group 1), BP with 808-nm NIR irradiation , aCD47 , BP with 808-nm NIR irradiation plus aCD47  and BP with 808-nm NIR irradiation plus aCD47 . The mice in groups 3, 4 and 5 were injected with aCD47 at a dose of 50 µg per mouse on day 5 for the second time. Body weight and tumour growth were then monitored in the following 3 weeks. There was no obvious influence on the body weights of the mice during the treatments  staining was performed. The histopathological analysis of lungs showed the presence of inflammation, hyperaemia, reduced alveolar volume and alveolar hyperplasia in the control groups , while the BP and aCD47 combined treatments (groups 4 and 5) resulted in reduced pathological abnormalities. Similarly, prominent oedema in the liver was observed in the control groups , but much less oedema was observed in the combination treatment groups (groups 4 and 5). In addition, lymphoma cell metastasis distributed in the spleen was observed in the control groups, whereas limited spleen metastasis was observed in the combination-treated groups. The above results suggest that BP and aCD47 combination treatments resulted in inhibition of spleen metastasis and promoted pathological damage in the A20 tumours. No obvious inflammation, lesions or histological changes were observed in the heart and kidneys in each group, indicating that various treatments cause low toxicity to the heart and kidney Fig. . Howevernts Fig. . In gene63. To examine the synergistic anticancer activity of a combination of BP-based PTT and aCD47 in more detail, we randomly allocated A20 tumour-bearing mice into four groups: group 1: untreated; group 2: BP with 808-nm NIR irradiation (20 µg of BP per mouse); group 3: aCD47 (50 µg of aCD47 per mouse); group 4: BP with 808-nm NIR irradiation plus aCD47 (20 µg of BP and 50 µg of aCD47 per mouse at 2-h post PPT treatment). The residual tumour tissues from these groups were harvested at 48-h post treatment. The M1-like macrophages in the tumour tissues were analysed by flow cytometry. The percentage of M1-type (CD80+CD11b+F4/80+) macrophages infiltrated in the residual tumours from the BP-based PPT and aCD47 combination treatment group was significantly higher than that in the control groups, suggesting that this combined therapy induced the polarisation of tumour-specific macrophages towards the M1 phenotype and thus altered the immunosuppressive status into a more permissive tumour microenvironment to promote tumour regression  can find cancer cells and then be stimulated to kill them directly65, while helper CD4+ T cells (CD4+) fight cancer cells indirectly by activating macrophages and cytotoxic T cells and secreting cytokines that regulate the immune response66. To further investigate whether this combination treatment boosts tumour-specific T-cell responses, we tested different T-cell populations in tumours after various treatments. Consistent with the above results, the absolute numbers of tumour-infiltrating lymphocytes  and CD8+ T cells were increased in the BP-based PPT group compared to the control group, suggesting robust TIL and CD8+ T-cell infiltration by hyperthermic ablation , which is activated downstream of the p38 MAPK pathways, was significantly higher compared with that of the control  signalling pathway is important for the regulation of numerous cellular processes. Dysregulation of MAPK signalling may result in tumour occurrence77. Since BP in combination with aCD47 therapy has the ability to trigger immune responses, we further examined the impact of combined treatment on a model of distant metastasis to determine whether local treatment could promote systemic immunity against cancer. A20 cancer cells were subcutaneously transplanted into both the left and right flanks of Balb/c mice to mimic metastatic cancer. The tumours on the right and left sides were defined as the primary and distal tumours, respectively. When the tumour volume reached ~100 mm3, the A20 tumour-bearing mice were randomly allocated into four groups as discussed in ‘Immune responses after BP-PTT plus aCD47 blockade’. First, the primary tumours were irradiated with an NIR laser  for groups 2 and 4. Subsequently, the mice in groups 3 and 4 were intratumourally injected with 50 µg of aCD47 at day 1 and day 5, whereas the left distal tumours were left untreated. As expected, the growth of primary tumours was inhibited by BP-based PTT and aCD47 treatment, but the distal tumours on the left side without treatment also showed pronounced regression, and five of the animals were macroscopically tumour free on day 21, achieving better efficacies than those of the other control groups . PEG-NH2 (5000 Da) was purchased from Nanocs, Inc. . Other reagents were analytical grade. Ultrapure water  was used for water-based dispersions.Liquid-phase exfoliation was used to exfoliate BP. Typical exfoliation was mainly divided into three steps: grinding, probe sonication and bath sonication. One hundred milligrams of bulk BP was dispersed in 1-methyl-2-pyrrolidone (NMP) (10 ml). Then, the bulk BP was ground by a hammer in NMP. After grinding, 90 ml of NMP was further added to the mixture. Then, the mixture underwent probe sonication with a power of 260 W for 8 h. To avoid overheating produced by probe sonication, we set the sonication process to an interval mode with an on/off cycle of 4/4 s, and the BP dispersion was immersed in ice water. After probe sonication, the BP dispersion underwent bath sonication for 12 h at a power of 300 W. The bath temperature was set to 10 °C.After these processes, appropriate BPNSs can be obtained and need to be selected from the mixture with bulk BP. Therefore, the resulting suspensions were centrifuged at 7000 rpm to remove the large BPNSs and bulk BP. The supernatant with the expected BPNSs was collected for further centrifugation at 17000 rpm for 30 min. Then, the precipitate was gathered and dried in a vacuum drying chamber. Then BPNSs were sealed in tubes and protected by the tinfoil package to avoid degradation. Then, the samples were stored in the refrigerator at 4 °C for further use.2 was mixed with BPNSs at a mass ratio of 2:1. After the sample was stirred for 2 h, the resulting dispersion was centrifuged through Amicon tubes  at 3000 rpm (4 °C) for ~20 min to remove the uncoated PEG-NH2 molecules and was further washed two times using the same centrifugation method. The pure PEG-modified BPNS sample was resuspended in PBS and stored in a 4 °C refrigerator for further use.For fabrication of PEG-modified BPNSs, PEG-NHhυ = 1486.7 eV, λ = 0.83 nm). Raman measurements were made on a HORIBA modular Raman spectrometer with 514-nm laser excitation. The UV–vis absorption spectrum was measured at nm employing a Cary 60 spectrometer from Agilent Company. A semiconductor diode laser, LSR808H , was used as the excitation source for conducting photothermal experiments. The measurement of photothermal experiments was carried out by using both a thermocouple and a thermal imaging camera (FLIR E-75).TEM (JEM1230) was used to measure the lateral size of the BPNSs, and AFM  was used to measure the thickness. For the TEM sample, the BPNS dispersion was placed on a copper grid and dried in an oven. The spin-coating method was used to prepare the AFM sample on a silicon substrate. Then, the high-resolution AFM images were scanned at 512 pixels in one line. HRTEM and SAED were performed on Tecnai G2 F30 equipment with a voltage of 300 kV. The XPS spectrum was acquired through a ULVAC PHI 5000 Versa Probe II with Al Kα , following the manufacturer’s instructions. All animal experiments were conducted following protocols approved by the Shenzhen International Institute of biomedical Research complied with all relevant ethical requirements.At 48 h after no treatment or the BP + aCD47 combined treatment, tumour slices were sampled and homogenised in the presence of cell lysis buffer, proteinase inhibitor and phosphatase inhibitor cocktail. The supernatants were collected, and phosphorylated proteins related to the MAPK pathway were analysed by a Human/Mouse MAPK Phosphorylation Array , following the manufacturer’s instructions.3 were randomly assigned to four groups and treated as described previously. At 48-h post treatment, the tumour slices were collected from the sacrificed mice for detection of the immune effect. Briefly, tumour slices were cut into small pieces and subjected to digestion by collagenase type IV , hyaluronidase  and DNase I  at 37 °C for 30 min with shaking. RBC lysis buffer  was used for the removal of red blood cells. Dead versus live cells were measured by a Zombie NIRTM Fixable Viability Kit , following the manufacturer’s instructions. The cells were stained by antibodies with fluorescence-labelled anti-CD3-APC , anti-CD8-FITC , anti-CD4-PE , anti-CD45-APC , anti-F4/80-FITC , anti-CD11b-PE/Cy7  and anti-CD80-PE , following the manufacturer’s instructions. CD3+CD8+ cells were defined as CTL. F4/80+CD11b+CD80+ cells were defined as M1 phenotype macrophages, and CD45+CD11b+ cells were defined as murine monocytes. The stained cells were measured on a DxFLEXF flow cytometer (Beckman Coulter), and data were analysed using FlowJo V10 software.For determination of the immunological effect of BP-aCD47 combination treatment, A20 tumour-bearing mice with a volume of ~100 mmt test for the comparisons of two independent groups, while one-way analysis of variance was used for comparisons of more than two groups by using GraphPad Prism software, and P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) and P < 0.0001 (****) denote statistical significance.Mean and standard error of the mean (s.e.m.) were used to show the data. Statistical analyses were performed using two-tailed unpaired Student’s http://www.nature.com/lsa).Supplementary information accompanies the manuscript on the Light: Science and Applications website (Supplementary Information


This work focuses on the effect of curdlan (CL) on dynamic viscoelastic property, thermal reversible property, viscosity, and the fluid types of hydroxypropyl methylcellulose (HPMC) at different temperatures. Compared to the blends at 25 °C, the blends had a smaller linear viscoelastic region (LVR), a higher gel strength, and larger storage modulus (G’) and loss modulus (G”) values at 82 °C. G’, G”, gel strength, and viscosity increased with the increase of CL. Repeated temperature sweep led to increased G’ and G” of HPMC/CL blends. For HC6 and HC8, the gel formation temperature of the repeated temperature sweep was significantly lower than that of the first sweep. The samples at 82 °C, except for the sample with 8% CL, were all yield-shear thinning fluids, and the samples at 40 °C were shear thinning fluids. The creation of HPMC/CL and its rheological research might provide some methodological references for the study of other thermal–thermal gel blends. Hydroxypropyl methylcellulose (HPMC) is a modified cellulose derivative that can be widely used in the food industry as pre-coatings for fried foods, edible film for food preservation, thickening agent, suspension agent, and slow-release material for food and medical usage, etc., due to its thermal gel, film-forming properties, thickening property, dispersibility, and solubility ,2,3,4. HAlcaligenes faecalis [Curdlan (CL), a linear triple helix polysaccharide, composed of one 3-β-linked D-glucose unit produced by a strain of faecalis , is a hofaecalis . Since ifaecalis . It is ifaecalis . Howeverfaecalis . In low-faecalis . Due to faecalis .So far, no document has been retrieved on the rheological and gel properties of HPMC/CL blends. This research intended to study the rheological property of thermal–thermal blending gels, including dynamic viscoelastic properties, viscosity, and fluid types at different temperatures, and thermal reversible properties. Furthermore, the morphology of the blends was studied, as well. The establishment of this thermal-thermal blending gel system and research on its rheological properties might provide some methodological and theoretical references for other thermal–thermal blending gels. This research might also provide some theoretical guidance for the application, such as a synergetic thickening agent or pre-coatings for fried foods of HPMC/CL blends.w/w) HPMC: 6 mPa.s; weight-average molecular weight (Mw): 2.441 × 104 g/mol) was bought from Hopetop Pharmaceutical Company . A food-grade CL  was purchased from Jiangsu Yiming Biological Technology Co., Ltd. .HPMC (ZW-E6)  HPMC, followed by the addition of CL to prepare the HPMC/CL system with 10% HPMC (w/w) and 0%, 2%, 4%, 6%, and 8% CL (wCL/w(water+HPMC)), respectively. Finally, the above suspensions were continuously stirred at 60 rpm for 1 h and at 40 rpm for another 1 h at room temperature before a rheological test. HC0, HC2, HC4, HC6, and HC8 were used to mark the five suspensions, respectively.HPMC was dispersed in deionized hot water (90 °C) by slowly agitating for 20 min, then it was cooled to room temperature by stirring at 30 rpm for 40 min to prepare 10% . The plate rotor PU40 SR1343 SS, with a diameter of 40 mm and a gap of 1.0 mm, was chosen for the strain sweep, frequency sweep, and temperature sweep, while the cylindrical rotor C25 SW1114 SS, with a diameter of 25 mm and a gap of 1.0 mm, was chosen for the shear rate sweep. Except for the temperature sweep, pretreating was done for all other sweeps. For pretreating, all samples were heated to 95 °C and maintained for 5 min in the sample holder; they were then cooled to 82 °C and 25 °C, respectively. The sweep began when the sample stabilized. All the samples tested at higher temperatures were sealed with silicone oil to prevent water evaporation. The specific methods are described below:The strain sweep was mainly carried out to obtain the linear viscoelastic region (LVR) ,21. For The frequency sweep was tested in the range of 0.1–10 Hz, with the tested strains of 0.1% at 25 °C and 82 °C, respectively.σ) and shear rate values (γ) into the corresponding fluid equations. The fluid equation corresponded to the shear thinning fluid and the yield-shear thinning fluid, as displayed below, respectively:K), the flow behavior index (n), and yield stress (yσ) can be calculated.The shear rate sweep was tested using the procedure of the Toolkit_V001–1 Table of Shear Rates/Equilibrium Flow Curve in the range of 1–500/s at 40 °C and 82 °C, respectively. The flow pattern could be ascertained by fitting the shear stress  at 10 kV.p < 0.05).SPSS Statistics 19  was used to analyze the data. The data were presented as the mean ± standard deviations (SDs). The mean was compared using one-way analysis of variance (ANOVA) followed by Duncan’s multiple comparison tests. Different superscript letters mean that values differed significantly  at 82 °C was larger, for example, with HC8 at 82 °C, it showed G’ of about 60 times larger than the blend at 25 °C. The reason for the tremendous changes of strength at 82 °C may be that HPMC gel is thermo-reversible, and that CL can form stronger gel structure at higher temperatures, and that there might be an inter-network between these two thermal gels. Compared with HPMC, the G’ and G” of the HPMC/CL blends increased with the increasing content of CL at both temperatures. The increase extent of the blends at the high temperature was higher than that at the low temperature, which may be because both CL and HPMC formed gel networks at high temperatures and co-gel may have been formed.The observed strong increase of both moduli with increasing CL content at both lower and higher temperatures in A temperature sweep of HPMC/CL blends was carried out twice, and the obtained sweep curves are illustrated in The effective molecular chain hardness, junction zone strength, and bond quantities contribute to G’ and friction energy consumption in a liquid state, which includes mobility, movement, friction of small molecules, and the vibration and rotation of functional groups all contribute to the value of G” ,26. ThesThe G’ and G” values of the samples in all the three stages increased with the increasing CL content. At a higher temperature, the G’ and G” of all the samples increased tremendously with the increasing CL content. The increasing extent was especially tremendous for HC8. At lower temperatures (lower than the gel formation temperature), the G’ and G” values of samples HC0, HC2, HC4, HC6, and HC8 were small, and the increasing extents with increasing CL content were also small, corresponding to their liquid properties at a lower temperature.From As shown in One thing to mention: for the second temperature sweep, HC6 and HC8 did not show gel properties at 25 °C, while the strain sweep and frequency sweep of HC6 and HC8 in n, K, σy, and r2 values of the HPMC with different CL contents at different temperatures are calculated and displayed in 2, meaning that they were all yield-shear thinning fluids, which are also recognized as Herschel-Bulkley fluids. For yield-shear thinning fluids, due to the attraction between intermolecular forces, hydrogen bonds, and hydrophobic forces, three-dimensional gel network structures can be formed at rest. These network structures are just like solids under low stress, which only produce elastic deformation and do not flow. Only when the external force exceeds a certain critical value, the gel network structure will be destroyed and the fluid will flow. The yσ value increased with the increasing CL content, with HC4 and HC6 showing significant increases, meaning that these two blends showed more obvious solid-like behavior. Meanwhile, Equation (1) can be used to fit the results of all the samples at 40 °C, and the fitting results are very well based on the value of r2, meaning that the samples at 40 °C were all shear thinning fluids. The n value of Newtonian fluid is 1, while, for shear thinning fluids, the n value is less than 1. The more the value deviates from 1, the clearer the shear thinning behavior [n value decreased and the K value increased, meaning that samples with more CL showed stronger shear thinning behavior and increased viscosity. With the increase of CL, the concentration of polymer increased, which increased the molecular chain density per unit volume and the probability of hydrogen and hydrophobic interactions between the molecules, resulting in a more complex arrangement of the samples. Meanwhile, after shearing, the interactions of molecules were destroyed, with the molecule chains being arranged simply and linearly along the direction of the shear stress. Samples with higher CL content were subjected to more disruption of molecular interactions after shearing, thus a stronger shearing thinning extent and larger decreasing extent of viscosity were observed for these samples.According to fluid Equations (1) or (2) in behavior . For samFor the samples with the same CL content, the viscosity of HPMC/CL blends were in the order: 82 °C > 40 °C. A much higher viscosity at a higher temperature was due to the gel structure formation. The shear thinning extent of samples also increased with the increasing temperature because gels at higher temperatures underwent much more disruption of molecular interactions than the solution at lower temperatures, since gels are compact network structures. The blends had higher gel strength and viscosity at higher temperatures and were anticipated to be used as thickening agents for hot drinks, skeletons for bakery products, and pre-coatings for fried foods as water retention scherm.The morphological structures of HPMC/CL blends incubated at 82 °C are shown in The establishment of this thermal–thermal HPMC/CL blending gel system and the research on its rheological properties provide some methodological and theoretical references for other thermal–thermal blending gels. The viscosity, moduli, gel strength, and compactness of the gel network of HPMC/CL blends at 82 °C were tremendously higher than that at a lower temperature, which provides some theoretical guidance for their application, such as, thickening agents for hot drinks, skeletons for bakery products, and pre-coatings for fried foods as water retention scherm.


Optofluidic devices are capable of detecting single molecules, but greater sensitivity and specificity is desired through hydrodynamic focusing (HDF). Three-dimensional (3D) hydrodynamic focusing was implemented in 10-μm scale microchannel cross-sections made with a single sacrificial layer. HDF is achieved using buffer fluid to sheath the sample fluid, requiring four fluid ports to operate by pressure driven flow. A low-pressure chamber, or pit, formed by etching into a substrate, enables volumetric flow ratio-induced focusing at a low flow velocity. The single layer design simplifies surface micromachining and improves device yield by 1.56 times over previous work. The focusing design was integrated with optical waveguides and used in order to analyze fluorescent signals from beads in fluid flow. The implementation of the focusing scheme was found to narrow the distribution of bead velocity and fluorescent signal, giving rise to 33% more consistent signal. Reservoir effects were observed at low operational vacuum pressures and a balance between optofluidic signal variance and intensity was achieved. The implementation of the design in optofluidic sensors will enable higher detection sensitivity and sample specificity. The optofluidic detection of particles in liquid waveguide channels has recently grown in significance ,4,5,6,7.The nature of the microfluidic channels found on optofluidic sensor platforms sets some of the ultimate sensitivity and accuracy limits. The small cross-sectional area of the channels typically means operation in the laminar flow regime, which results in a parabolic velocity distribution. Fluid that is in the center of the channel flows faster than fluid at the walls. This means fluorescent particles, being distributed evenly through a channel, would spend different amounts of time in perpendicular excitation light beams as they flow past excitation points introducing variation in excitation times and fluorescent signal generation. Additionally, the excitation light exhibits an optical mode intensity profile that results in a distribution of generated fluorescence intensities. Additionally, the collection efficiency of fluorescence in a liquid-core waveguide is dependent on particle position ,16. PartHydrodynamic focusing (HDF) promises to enhance the detection capabilities in optofluidic channels by controlling the position of target particles in the fluid channel. This results in narrower velocity and excitation intensity distributions, which means less variation in the fluorescent signal intensity distribution . Three-dHDF is commonly achieved using buffer fluid or sheath flows to surround and squeeze a sample fluid stream. The engineering challenge comes in designing and fabricating the intersection for these flows. Two-dimensional hydrodynamic focusing is relatively straightforward, even at the 10-μm scale. It simply requires flowing buffer fluid from the left and right sides of the sample stream to achieve horizontal, or in-plane, focusing, as in A wide variety of interesting designs have been developed to perform 3DHDF while using buffer fluid at the microscale. One existing method literally injects sample fluid into a buffer fluid stream using a nanoneedle, or micronozzle . The micSimpler, “single layer” designs make use of clever fluid dynamics properties to induce 3DHDF with buffer fluid through secondary flow production. This takes the form of Dean Vortex generation in fast moving fluids. The vortices induce flow orthogonal to the main flow direction, drawing out and shaping the sample stream. The microscale fluid channel structures can be simpler because the fluid dynamics do some of the work, reducing complexity in fabrication and operation.The curve design, sometimes called “microfluidic drifting”, requires four or five fluid reservoirs to operate . Fluid mThe contraction-expansion array design (CEA) works in a similar manner, inducing secondary flow in the form of Dean Vortex generation . Rather The last of the single-layer, vortex generator designs discussed here is the microstructure stream-sculpting design . More coNote that these designs are more typical at the 100-μm scale or larger. When shrunk to the 10-μm scale, the fluidic resistance of the small cross-section fluid channels dramatically increases and the flow velocities that are required to induce the secondary flow focusing effect become impractical due to the massive microfluidic channel backpressures . Thus, lWe present a 3DHDF design requiring just one layer of sacrificial material. The low-pressure chamber design, or pit, requires four fluid ports to operate and it promises improved device yield over multi-layer designs. Moreover, it operates by pressure driven volumetric flow ratio, enabling low velocity focusing in channels with dimensions around 10 µm. We will present computer simulations, fabrication processes, and optical characterization of the focusing completed in microscale channels. Fluorescent beads in a flowing aqueous solution are used to characterize the design and demonstrate the enhanced detection capabilities of the system. We have previously reported 3DHDF at the 10-μm scale while using a volumetric flow ratio technique . HoweverQuantifying fabrication complexity is challenging, as it includes processing time and difficulty. For example, multi-layer photoresist designs are extremely difficult in that subsequent layers must be perfectly processed on the first try, whereas single-layer designs allow for layer stripping and repatterning. Additionally, each layer attempt requires between three and six hours. Moreover, multi-layer designs prove to be less robust, resulting in low device yields. Every parallel processed device can become simultaneously damaged near the end of the multi-week fabrication process, requiring complete device remanufacturing. Moving to a single-layer design greatly simplifies the fluid volume formation process. The schematics presented in 2. Four fluid reservoirs manage the sample, buffer, and waste fluid on the chip. The fluid on the chip flows from left to right, with the buffer fluid coming in contact with the sample fluid near the center of the chip at the focusing junction. The fluid focusing occurs before the liquid-core waveguide fluid channel is intersected by solid-core waveguides that carry light for fluorescence excitation and signal collection. This intersection point is called the excitation region, or excitation volume when referring to the fluid.Q/Q) while using the known hydrodynamic relation P = QR or Q = P/R, where P is pressure, Q is volumetric flow rate, and R is fluid resistance. This relation is derived from Newton’s second law, to the Navier–Stokes equations, to the Hagen–Poiseuille relation. The relation assumes incompressible laminar Newtonian fluid flow through a circular pipe. The hydraulic radius is substituted to apply the relation to a rectangular duct. The pair of volumetric flow ratios solved were of the buffer fluid flow and sample fluid flow, and of the buffer fluid flow and the outlet fluid flow. The pressure drop was chosen to be approximately equal between the ports and the fluid focusing junction, thus canceling out and leaving just the R terms. The R terms were expanded into the Hagen–Poiseuille form, the constants canceled out, and the hydraulic radius substituted, leavingL is fluid channel length, w is fluid channel width, and h is fluid channel height. Subscript 2 indicates terms relating to the buffer fluid channel and subscript 1 indicates terms that relate to the sample (or outlet) fluid channel. This equation was input into an online graphical calculator, called Desmos , which allowed for us to keep track of parameters and view the solution visually. Each Q ratio value was input manually as calculated from the input velocities and cross-sectional areas of the inlets from the CAD model (Q = VA). The ratio was subtracted from both sides of the equation to create the slope intercept form for graphing. One of the dimension variables in the equation was replaced with the variable x to act as the solution. The equation was multiplied by a large number in order to cause the graphed line to appear nearly vertical for ease of dimension determination. The variable values and solution variable were adjusted until the solutions were deemed as acceptable for fabrication tolerances.The fluid channel dimensions between the focusing junction at chip center and the fluid ports were determined by solving a pair of volumetric flow ratios . The photonic signal passed through a penta-bandpass optical filter  before it was detected by a Time-Correlated Single Photon Counting (TCSPC) avalanche photodiode (APD)  and analyzed using our event detector scripts developed in Python and available on github  . It utilizes continuous wavelet transform (CWT) with custom made wavelets to enhance the signal in both time and scale domains, followed by the threshold method to localized events accurately in time and scale. Scale information is designed to match with the Δt value that was related to subsequent bright spots for a given multi-peak signal  diluted to 107/mL concentration to elicit discrete signal peaks for analysis. The laser light is coupled to the multi-mode interference excitation waveguide (MMI), which was designed to generate a 75 μm long multi-spot illumination pattern at the intersecting liquid-core waveguide, as seen in The same apparatus, as shown in −8 cm2/s, two orders of magnitude lower than the dye [The lateral spreading of molecules in a focused stream is approximately proportional to the square root of diffusivity. The beads have a reported diffusivity of about 2 × 10 the dye ,37. Two-The effect of 3DHDF on fluorescent signal quality was measured by first filling all of the fluid channels with a fluorescent bead solution as a control test. In this case, there is no effective focusing of a particle stream because the entire cross section of a fluid channel is filled with particles. Measured fluorescence for this case was then compared to the case, where the sample fluid channel was filled with solution containing beads and the buffer channels with water. In both cases, fluid was drawn through the optofluidic channels with negative pressure at the outlet and illuminated with multiple spots of 633 nm laser light through the solid-core MMI waveguide. An avalanche photodiode at the chip edge collected the fluorescent signal captured orthogonally in the liquid-core waveguide and a computer reported the intensity in counts per 0.1 ms, as observed in the traces in These plots show the measured optical intensity versus time, with intensity spikes representing fluorescing beads passing through an excitation point. When comparing the control trace to the experiment trace we see an almost sevenfold decrease in events captured per time. This is likely due to the buffer fluid in the 3DHDF case occupying a large fraction of the channel volume and reducing the number of beads passing through the channel per time. An improvement in detection performance can be represented by the coefficient of variance (CV) for the measured signal. This is calculated by dividing the standard deviation of the signal intensity by the mean of the distribution. A smaller CV represents a narrowing of the distribution or an increase in mean. They both indicate enhanced detection of optofluidic sensors promised by HDF. The data found in We wanted to test how the mean and standard deviation of signal traces that were collected at various vacuum pressures would relate. Increasing the vacuum pressure results in a greater pulling force on the fluid through the channels, and thus higher fluid velocities and a lower chance of detecting all emitted photons. This is related to the time resolution and dead time of the detector. Fluorescence happens on the nanosecond scale, which means the beads should have enough time to be excited and emit fluorescent signal, but the detector is unable to detect all of the received photons in the short time interval that the fluorophore spends in the excitation volume. The result is lower signal intensities. We changed the pressure in increments of 5 from −5 inHg to −25 inHg as well as −28 inHg, the limit of the vacuum system. We concluded that a minimum of −15 inHg vacuum pressure should be used to overcome the reservoir effects that were observed in the testing of the device. This finding, in conjunction with the signal intensity data found in We compared the device yield of the silicon-based microchips in over 300 total devices. The present design showed an average of 45% yield when compared to the average 29% yield of the previous design, a 1.56 times improvement in device yield. We attribute this yield improvement to the robust design while using a single-layer of photoresist to form the fluid channels. A 10-μm scale, single layer 3D hydrodynamic focusing design was implemented, which improved device yield by 1.56 times over previous work. The low-pressure chamber, or pit, is the key feature that induces 3D focusing using buffer fluid that is delivered by negative pressure-driven flow, controlling the position of target particles in the fluid channel during fluorescent signal generation. This mitigates the disadvantageous natural phenomena that are present in microfluidic channels by narrowing signal intensity and flow velocity distributions and constraining target particles to highest collection efficiency regions. It does this while using just four fluid reservoirs. The concept, modeling, fabrication, and testing were outlined. Fluorescent beads representing molecules of interest were analyzed to find 33% more consistent signal with focusing. The reservoir effects were observed at low operational vacuum pressures and an optimal detection scheme was used to maximize signal consistency while maintaining high signal intensity. Signal intensity and flow velocity coefficients of variance matched expectations by decreasing between the control and the experiment, indicating successful focusing and detection enhancement. Further improvement and optimization in signal intensity can be achieved by aligning focused particle streams with the optical excitation mode center and with collection modes in the liquid-core waveguide. This could be achieved with the current geometry while using independent fluid inlet controls to flow additional buffer fluid volume. A gravity driven back-pressure method would be ineffective at higher operational vacuum pressures as the effect becomes dominated. Alternatively, the pit approach could be redesigned to efficiently operate with negative pressure-driven flow.


Pericardial constriction is one of the complications of purulent pericarditis (PP). Most difficult to treat, which may develop both in early and in the late period of the disease, resulting in a very poor prognosis.We present case series of 4 patients with purulent pericarditis, in whom direct intrapericardial administration of recombinant tissue plasminogen activator (r-tPA) was used.Management of PP requires a combined surgical and medical approach. The most important is complete drainage of the effusion by subxiphoid pericardiotomy connected with complementary use of broad-spectrum antibiotics. Despite the use of broad- spectrum antibiotics, in some patients a large volume of daily drainage is still present. Constrictive pericarditis as a complication of PP is observed in majority of patients.Intrapericardial administration of fibrinolytic agents, although not strongly recommended, can improve efficacy of antibiotic treatment especially in patients with loculation fluid and can prevent the development of constrictive pericarditis.r-tPA was applied at a dose of 20 mg dissolved in 100 ml of normal saline in a 100 ml syringe, administered by a large pericardial drain (Pezzer drain) installed into the pericardial cavity during pericardioscopy. The tube was closed and re-opened after 24 h.No serious complications, such as bleeding, allergy or hypotension, were noted.We present case series of 4 patients with purulent pericarditis, in whom direct intrapericardial administration of recombinant tissue plasminogen activator (r-tPA), prevented the development of constrictive pericarditis, and increased efficacy of antibiotic treatment without any significant complications. Purulent pericarditis (PP) is a rare cause of pericardial disease, with the prevalence of less than 3%, but with potentially life-threatening complication . Most frThe recommended treatment of PP is based on pericardial drainage and intravenous antibiotics administration .Intrapericardial use of fibrinolytic agents such as Streptokinase (SK) or Urokinase (UK) is suggested in case of early signs of pericardial constriction, fibrin deposits and loculation of the fluid .Despite proper multidirectional treatment, PP still has a 20–30% mortality rate . Late moClinical observations based on a small group of patients suggest that the intrapericardial administration of fibrinolytic drugs, although not strongly recommended, is an effective way to prevent the development of pericardial constriction and thus protects the patient from a pericardiectomy , 3. Up tWe present 4 patients with purulent pericarditis, successfully treated with intrapericardial administration of recombinant tissue plasminogen activator.The clinical data of 4 patients with confirmed PP, who were treated with intrapericardial recombinant tissue plasminogen activator (r-tPA) is presented in Table PP was recognized in the patients who were suffering from generalized weakness, dyspnoea, fever, chest pain and - in one case- septic shock. Pericardial effusion (hyperechogenic fluid around the whole heart and in some cases with a large amount of fibrin) was diagnosed by echocardiography. The purulent character of the fluid was confirmed by physical and biochemical examination .In one patient purulent pericarditis developed in the course of bacterial knee -joint inflammation, in another, diagnosed with common variable immunodeficiency, in the course of pneumonia and empyema. In the remaining two patients, the cause of pericardial infection has not been identified.S. epidermidis in another. Because of the fact that S.epidermidis was diagnosed in two different fluid cultures, it was considered a cause of purulent pericarditis and not a contamination. In the other two patients, the cultures of pericardial fluid were negative, probably due to previous antibiotic therapy.The purulent pericardial fluid was obtained from all of the patients, the cultures of the fluid tested positive for MSSA in one patient and for Subxiphoid pericardiotomy under general anesthesia has been performed in all of the patients with implantation of a large tube drain (Pezzer drain). The indications for intrapericardial fibrinolytic treatment (IFT) were: prolonged purulent drainage and fibrin deposits in pericardium (4 patients), early echocardiographic signs of pericardial constriction despite optimal treatment (3 patients), excessive pericardial drainage with signs of pericardial fluid inoculation (1 patient- case 4). Early echocardiographic signs of constriction were defined as: thickening of pericardium, the mitral inflow velocities showing a pseudo-normal or restrictive filling pattern (E/A > 1 and deceleration time < 150 ms), the respiratory variation in filling velocities, inferior vena cava plethora with expiratory hepatic vein diastolic reversal wave.r-tPA was applied at a dose of 20 mg dissolved in 100 ml of normal saline in a 100 ml syringe, administered by a large pericardial drain (Pezzer drain). The tube was closed and re-opened after 24 h.Two patients received a the single dose of r-tPA, and the remaining two – two doses.In one case (case 2) it was related to an extensive leak of pericardial fluid next to the drain, and the necessity of early opening of the drain, in another one (case 4) – with the persistence of a large amount of fibrin in the pericardium with fluid loculations. The repeated intrapericardial fibrinolysis in this patients was caused by excessive pericardial drainage.The treatment has been effective in all of the patients, diminishing the echocardiographic signs of early constriction in 3 patients, and reducing large pericardial drainage in the remaining one. The drains were removed 6–8 days after the last dose of r-tPA.The decision to remove the drain from the pericardium was made based on the reduction of daily drainage to less than 20 ml, and echocardiographic signs of remission .In each of these four cases, the successful treatment of purulent pericarditis was achieved.Chest computed tomography scans before and after intrapericardial r-tPA therapy in the patient with purulent pericarditis in the course of pneumonia and empyema due to common variable immunodeficiency syndrome were presented in Figs. An MRI was not performed due to the lack of availability and long duration of the study -two patients were in severe condition, one was in septic shock.Additionally MRI is not listed in 2015 guidelines as a first line diagnostic method for patients with PP.It is a valuable procedure to confirm constrictive pericarditis for stable patients, before surgery-pericardiectomy. All cases were monitored by bedside echocardiography every day.No serious complications, such as bleeding, allergy or hypotension, were noted. In one case, due to extensive leak of pericardial fluid next to the drain, it was reopened after 5 h.An appropriate, broad-spectrum antibiotic therapy was used intravenously, on average – for 21 days.In all of the patients- intrapericardial administration of fibrinolytic agents, caused remission of early sings of pericardial constriction, and regression of pericardial effusion.The patients were observed for 10 months (case 1), 13 months (case 2), 30 months (case 3) and 7 years (case 4) respectively. The repeated echocardiographic examinations revealed no signs of pericardial constriction, at follow –ups.We presented 4 cases of purulent pericarditis, treated successfully with intrapericardial r-tPA.Management of PP requires a combined surgical and medical approach. The most important is complete drainage of the effusion combined with complementary use of broad-spectrum antibiotics. Because of loculations and adhesions simple evacuation by surgical or percutaneous drainage may not prevent constrictive pericarditis . Open suThe guidelines of the European Society of Cardiology from both 2004 and 2015 recommend intrapericardial use of fibrinolytic agents in the therapy of PP, however the class of recommendations is low (IIa) as well as the level of scientific evidence (C) , 5. MoreAs PP is a rare entity, the randomized clinical trials are lacking and all the information concerning treatment efficacy and safety comes from case reports or case series.The clinical experience with IFT has been summarized by Augustin et al. in 2011 . MajoritLong term effect has been achieved in 86–89% of patients , 4. SeveOur own experience with SK used as IFT, concerns 4 cases, in one of them severe intrapericardial bleeding occurred as a complication, and in one case the therapy was ineffective and the patient received r-tPA as second line treatment (case 4) , 12. MajIn comparison to the above mentioned data from the literature , 4, the The safety of intrapericardial r-tPA was high, no complications have been observed in our patients, apart from a pericardial leak in one case probably due to the large volume of purulent fluid released from loculations.Use of r-tPA allows subsequent administration of the same drug , because of no immunization.Another important aspect concerns the timing of IFT in case of early constriction. As fibrin formation occurs during the first week of PP, fibrinolytic therapy should be used early, preferably in the first two weeks of the disease, however there in no consensus on the appropriate timing of fibrinolysis . It shouUse of r-tPA significantly decreased signs of constrictive pericarditis and decreased necessity of surgical treatment in cases with PP .Moreover, in the literature, there is no consensus on what dose and how intrapericardial fibrinolysis should be administered.The limited data available which is based on individual case reports present a range of doses from 2 mg/ 10 ml or 30 mg in 1 dose [Moreover, limited data concerning r-tPA dose for the treatment of empyema suggested a dose of 10 mg , 15.In our schema, based on the 4 cases, we proposed 20 mg r-tPA diluted in 100 ml normal saline and clamping the drain for 24 h.Without prejudice to the efficacy and safety of direct, intrapericardial administration of fibrinolytic agents in cases with confirmed PP, we would like to recommend, the described above schema consisting of 20 mg of r-tPA dissolved in 100 ml of saline in patients with previously performed subxiphoid pericardiostomy followed by insertion of the large drain connected with broad-spectrum antibiotics. We have included our recommendations in algorithm. Such therapy seems to be safe and effective in case of early pericardial constriction in the course of PP.Additional file 1. Algorithm.


We prove that if a data language and its complement are both recognized by nondeterministic register automata (without guessing), then they are also recognized by deterministic ones.


NAMPT knock-out mice Since the discovery of nicotinamide phosphoribosyltransferase (NAMPT) as a pre-B cell-enhancing factor in 1994 by Samal et al. in vitro and in vivo, phase I clinical trials with the first NAMPTi  in haematological or solid malignancies, showed no objective tumour remission, and toxicity. Besides the pharmacological properties of these drugs and their potential toxicity, one of the main aspects leading to the partial failure of NAMPTi treatment was due to the concomitant expression of others NAD-biosynthetic enzymes that can overcome NAMPT inhibition. It is now clear in the field that it is necessary to select tumours uniquely addicted to NAMPT activity in the generation of NAD. Novel NAMPTi are currently in pre-clinical and phase I-II clinical trials, highlighting an intense pharmacological effort to target this enzyme It has been shown that tumours require increased levels of energetic molecules. Metabolic rewiring is now considered one of the hallmarks of tumours, sustaining the production of cellular energy , and their growth and invasive capacities EBioMedicinein vivo model of PCa invasion. The regulation of NAMPT transcriptional expression and the activity of eNAMPT in promoting a tumour invasive phenotype are known in a variety of tumour types. However, two novel and significant findings emerged from this paper.The initial rational to develop NAMPTi was based solely on the role of NAMPT as an intracellular NAD-biosynthetic enzyme. However, NAMPT has emerged as a mediator of inflammation with important, context-dependent, extracellular functions The first one is that the elevated levels of eNAMPT in PCa have a predictive value for PCa diagnosis and correlate with aggressive PCa, suggesting that this protein could be consider a novel biomarker in PCa. This finding opens up the possibility of a clinically-utilized eNAMPT biomarker screen to assess PCa risk. To-date, prostate-specific antigen (PSA) levels are considered the only PCa biomarker to discriminate between prostate benign hyperplasia or inflammation and PCa. However, PSA assays, as discussed by the authors, suffer from high false-positive results, high variability, and fails to correlate with PCa aggressiveness. On the contrary, at least in the cohort analyzed in the paper, high (>30 ng/ml) eNAMPT levels are associated with aggressive and invasive PCa. Of course, future studies to confirm these results are needed. However, this paper proposes, for the first time, eNAMPT as a biomarker in PCa patients, to be used alone or in combination with PSA.in vivo, demonstrating that mice treated with polyclonal neutralizing anti-eNAMPT antibody had fewer and smaller colonies on the inferior diaphragm, confirming an inhibition of invasive capacity. The general idea of targeting eNAMPT in tumours is increasing, to counteract the extracellular functions of this protein, mainly linked to the activation of TLR4 and modulation of immune responses. It would be very interesting to address whether the block of eNAMPT acts on the PCa tumour as well as on the PCa tumour microenvironment. This approach might not be restricted to PCa, and perhaps a combination of pharmacological NAMPTi and eNAMPT blocking antibody to affect the dual roles of this enzyme/cytokine, will be a successful approach to halt tumour progression and instigate a less supportive tumour microenvironment.The second important and translational message is the therapeutic potential of eNAMPT neutralization, using a specific blocking antibody, to prevent the invasiveness and metastasis of PCa. The authors studied the capacity of PCa cells to invade the inferior diaphragmatic muscle V.A. contributed to the literature search, data analysis, data interpretation, and writing.Dr. Audrito has no conflicts of interest to disclose.