ALG13 c.320A>G mutation. Contrary to other known variants of type I CDGs, where transferrin isoelectric focusing revealed notably increased asialo- and disialotransferrin fractions, a normal glycosylation pattern was observed in the probands. To verify this data and give novel insight into this variant, we modeled the human Alg13 protein and analyzed the dynamics of the apo structure and the complex with the UDP-GlcNAc substrate. We also modeled the Alg13-Alg14 heterodimer and ran multiple simulations of the complex in the presence of the substrate. Finally, we proposed a plausible complex formation mechanism.Congenital Disorders of Glycosylation (CDG) are multisystemic metabolic disorders showing highly heterogeneous clinical presentation, molecular etiology, and laboratory results. Here, we present different transferrin isoform patterns (obtained by isoelectric focusing) from three female patients harboring the  Congenital Disorders of Glycosylation (CDG) are multisystemic metabolic disorders caused by defects in N-linked, or O-linked oligosaccharides, shared substrates, glycophosphatidylinositol (GPI) anchors, or dolichols pathways . There aALG13 gene (Asparagine-linked glycosylation homolog 13) dysfunction is related to type I CDGs. The product of the ALG13 gene, the Alg13 protein, is involved in the N-glycosylation process. Several mutations of the ALG13 gene have been reported, related mostly to epilepsy [epilepsy ,5,6,7, bepilepsy . Consideepilepsy ,10. Yet,The Alg13 protein is a highly conserved X-linked uridine diphosphate (UDP)-N-acetylglucosaminyltransferase required for the transfer of N-acetylglucosamine (GlcNAc) onto the extending lipid-linked oligosaccharide (LLO) structure, dolichol-P-P GlcNAc . This eaIn early 2005, Chantret et al.  suggesteN-acetylglucosamine-1-phosphate transferase (GPT) encoded by DPAGT1. It was reported that deficiencies of DPAGT1 are also related to a group of CDG disorders (formerly Ij type) [Protein N-glycosylation is a two-step process: it starts with the transfer of an oligosaccharide from a lipid-linked oligosaccharide onto proteins and is initiated by UDP-GlcNAc:dolichol phosphate Ij type) . The secDPAGT1 gene, the GPT enzyme, is mostly membrane-bound in the ER. For example, the hamster GPT enzyme, which was examined by Dan et al. [DPAGT1 gene is called Alg7 in S. cerevisiae [There is a high sequence similarity between the prokaryotic and eukaryotic enzymes involved in the first step of N-glycosylation . The pron et al. , consistn et al. , while sn et al. . It shourevisiae , and itsrevisiae , as wellrevisiae , who werrevisiae .Taken together all the above mentioned information, a clear mechanism appears; however, it still requires experimental confirmation. N-glycosylation is a process which takes place on the endoplasmic reticulum, and in the case of eukaryotes requires the presence of three enzymes, Alg7, Alg13, and Alg14, which can create a complex together. They may share a common binding site cavity, or the cavity is separated by some flexible regions, which act as molecular gates . Alg7 isALG13 dysfunctions, where transferrin isoelectric focusing (IEF) revealed notably increased asialo- and disialotransferrin fractions, in the patients harboring the p.Asn107Ser mutation, a normal glycosylation pattern was observed , similarly to mass spectrometry (MS) results, previously described in one individual [ALG13 variant. We complemented the report by an exhaustive homology modeling study of the human Alg13 protein and the complex formation with Alg14. We ran in total 6 µs of molecular dynamics (MD) simulations to examine the behavior of the Alg13 protein, its complex with the UDP-GlcNAc substrate and the Alg13-Alg14 heterodimer in the presence of the substrate. We identified a conformational change which is crucial for the substrate transport and heterodimer formation that was not observed in the Asn107Ser variant of the human Alg13 protein. We hope that our results shed light on the role of the human Alg13 protein.Contrary to other known variants related to dividual . There idividual . Since tALG13 gene were described previously by Paprocka et al. [Three female patients with a de novo c.320A>G variant in the a et al. . In all Anthropometric measurements were undertaken for Patients 1 and 2. A Wolański liberometer (a type of an infantometer accurate to 1 mm) was used to measure the supine length of Patient 1. A stadiometer (accurate to 1 mm) was used to measure the standing height of Patient 2. Their weight was determined using an electronic scale accurate to 0.05 kg. A non-stretchable tape was used to measure their head and chest circumferences (accurate to 5 mm). Anthropometric measurements were obtained for each patient  followinThe age and sex data were standardized based on the mean and standard deviation of the examined feature in a given age group of the healthy children using Polish reference charts ,24,25.The transferrin isoforms were examined by IEF in agarose gel on a Multiphore 2117 apparatus (LKB). The serum transferrin was saturated with iron by the addition of ferric citrate and sodium bicarbonate. The iron-saturated transferrin was electrophoretically separated into fractions using a pH gradient of 5–7 formed by the ampholines added to the 1% agarose gel. The position of the isoforms in the gel depended on the amount of electronegative sialic acid residues on the glycans associated with each isoform. After separation, the isoforms were precipitated into the gel with specific polyclonal-rabbit anti-human transferrin antibodies. Proteins that were not bound by antibodies were washed away from the gel. The gel was dried, and the protein complexes were stained within a 0.5% Coomassie Brilliant Blue solution. The percentage of individual fractions were determined using an Appraise densitometer (Beckman). The procedure was based on the method described by Van Eijk et al.  with ourCE was used for detection of CDT—Carbohydrate Deficient Transferrin  . The CAPALG13-is2 isoform of human Alg13 protein (UniProt ID: Q9NP73).The Alg13 homology model was created by the I-TASSER webserver  using thEscherichia coli MurG in a complex with UDP-GlcNAc (PDB ID: 1nlm) and the Pseudomonas aeruginosa MurG structure in complex with UDP-GlcNAc as templates. 10,000 homology models were created using the automodel class implemented in Modeller 9v19 [P. aeruginosa MurG structure, and the best model was selected based on the lowest DOPE score.The selected models of the Alg13 protein and the Alg14 structure downloaded from the SWISS-MODEL repository (ID: Q96F25)  were useler 9v19 . The subThe quality of all used models, namely the Alg13 homology model, the Alg14 structure, and the dimer model were evaluated by ProSA  and PROC−1 × Å−2), and a final minimization with no constraints of conjugate gradient energy minimization. Next, a gradual heating was performed from 0 K to 300 K over 20 ps using a Langevin thermostat with a collision frequency of 1.0 ps−1 in periodic boundary conditions with constant volume. The equilibration stage was run using the periodic boundary conditions with a constant pressure for 1 ns with 1 fs step using Langevin dynamics with a collision frequency of 1 ps−1 to maintain the temperature. The production stage was run for 200 ns in five repetitions for all models, with a 2 fs time step using Langevin dynamics with a collision frequency of 1 ps−1 to maintain a constant temperature. The long-range electrostatic interactions were modeled using the particle mesh Ewald method with a non-bonded cut-off of 10 Å, and the SHAKE algorithm was used to maintain the geometrical constraints. The coordinates were saved at an interval of 1 ps.The H++ server  was usedThe MD simulations were processed with the Molecular Mechanics Generalized Born Surface Area (MMGBSA) and the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) implemented in AMBER 18 suite  to obtaiFoldX suite  was usedThe PredictSNP  server wThis section is divided into experimental and computational parts. The first part presents the transferrin isoform patterns of our three patients obtained by IEF. We observed differences in the transferrin isoform patterns between our patients, as well as between samples taken from the same patient at different times. These observations have led us to search for differences in the mechanism of action between the native and Asn107Ser variants of the Alg13 protein. Therefore, the experimental part is complemented by the computational part. The latter describes the homology modeling of human Alg13 protein, the molecular dynamics simulations of the apo (“empty”) structure of Alg13, and its complex with the UDP-GlcNAc substrate. We also created an Alg13-Alg14 complex homology model based on bacterial MurG proteins with a bound substrate, and finally we ran MD simulations of the Alg13-Alg14 heterodimer in the presence of the substrate.For detailed descriptions of the patients, please refer to our previous article by Paprocka et al. . Here weDuring the latest assessment, Patient 1 was 3 years and 8 months of age. She attended a kindergarten for children with special needs, to which she adapted very well. She needed foot orthoses, but was able to walk independently with some coordination issues. She has a broad-based gait with uplifted forearms. Her speech was still severely delayed, she could say only “mama”. Seizures were not observed and antiepileptic drugs had been reduced.Patient 2, now 6 years old, started to walk independently at the age of four, and it is no longer a wide-based gait but more natural. She is continuously rehabilitated and undergoes special education with a psychologist, neurologist, SI therapist, hand therapy, and group social therapy. There are still problems with eating—she often chokes, does not eat on her own (cannot use a spoon/fork), and cannot drink from a cup despite constant training at home and in therapy classes—she drinks from a bottle. The patient still does not speak, and her imitation ability is very limited. For two years, she has had alternative communication introduced. When asked, she can often show the correct answer (but only those she is regularly shown and told); she distinguishes between symbols yes/no and often shows adequately to the situation. Unfortunately, the proband understands very little, can only react to her name, and addresses regularly repeated requests such as “hands up”. She does not report basic physiological needs and is still diapered. As for the patient’s medications, she is still on Sabril and Lamitrin S . Epileptic seizures occur only during sleep, but they are quite frequent—every second/third night. They appear as follows: the patient wakes up during the night, and for a few minutes, she periodically blinks her eyes and tenses her body, after which she falls asleep. Seizures very rarely require Relsed infusion. The patient has been attending a private kindergarten for the second year now. Although the progress is slow, she is very eager to participate in the classes and even demands continuing when they are over. The patient has a very gentle and cheerful disposition, smiles often and loves to cuddle. She is very friendly; she demonstrates many behaviors on the autism spectrum, but the diagnosis has not been made.Patient 3, aged 7 years and 9 months, is the most severely delayed. She is wheelchaired, cannot speak and suffers from everyday epileptic attacks, especially during bedtime. As the patient cannot swallow solid pieces, she only eats liquid food. Her diet is high in carbohydrates, mainly potatoes, and therefore a ketogenic diet is not possible.Anthropometric measures were undertaken for Patients 1 and 2, while Patient 3 was unavailable. The results were then standardized, based on the mean and the standard deviation of the examined feature in a given age group of healthy Polish children. The resulting z-scores for Patient 1 and Patient 2 are shown in To determine the transferrin pattern, we used IEF. For each patient we ran two analyses at different times. Patient 1 showed a normal transferrin pattern in both trials. Patients 2 and 3 displayed abnormalities in at least one trial. Patient 2 displayed a slight elevation of asialo-, monosialo-, and disialo fraction in the first trial, while in the second trial the pattern was normal. Patient 3 displayed a normal pattern in the first trial, but in the second trial we observed a slight elevation in only the disialo- fraction. The results are summarized in In light of the available publications on the subject, these results seemed surprising. Therefore, we also analyzed samples using another method (CE), which confirmed the results . PatientALG13-is2 isoform of human Alg13 protein. These two factors can substantially impact the quality of the hAlg13_hm model. Both Saccharomyces cerevisiae Alg13 models were obtained as a set of ensembles, so for our analysis we used only the structure of the first ensemble . According to the results of bioinformatic analyses using the PredictSNP webserver to assess the effect of the Asn107Ser mutation, the mutation is deleterious, although the protein is produced . HoweverTo investigate the behavior and effect of the introduced mutation on the human Alg13 protein, we ran multiple repetitions of molecular dynamics simulations of the previously built model and the model with the introduced Asn107Ser mutation. To describe the simulations’ results, two commonly used metrics were used, RMSD (Root Mean Square Deviation) and RMSF (Root Mean Square Fluctuation), to measure the quality of each simulation and the model itself. RMSD measures the overall protein stability; it is used to detect conformational changes. RMSF provides information on the average flexibility of a single residue during the simulation time. RMSD and RMSF for the native protein and mutant variant are shown in Human Alg13 protein is relatively stable. The RMSD plots of the native Alg13 structure fluctuated around 4–5 Å and suggested that the protein does not undergo rapid conformational changes. On the contrary, the RMSD plots of the Asn107Ser variant substantial and rapid conformational changes. In three out of five simulations the RMSD values exceeded 8 Å, while for the two other simulations they were fluctuating near 6 Å . HoweverWe then measured the distance between the starting position of residue’s 107 Cα atom and its position during the whole simulation time . The AsnP. aeruginosa MurG protein (PDB ID: 3s2u).To investigate the influence of the UDP-GlcNAc substrate on the Alg13 protein behavior, we ran five repetitions of the native Alg13and the Asn107Ser variant complexed with UDP-GlcNAc. The position of the substrate was modeled according to the similar crystal structure of the The human Alg13 protein’s complex with the substrate was relatively stable in both the native protein and the Asn107Ser variant. Interestingly, the RSMD and RMSF plots of the native Alg13 suggest that in the presence of the substrate the protein gained flexibility in the C-terminal and the region comprising the α3 helix and β3 strand . On the Using MMPBSA and MMGBSA approaches we analyzed the free energy of the substrate binding in both the native and the Asn107Ser variant of the human Alg13 protein . The resE. coli MurG and P. aeruginosa MurG. The position of the substrate was modeled according to the P. aeruginosa MurG which had better resolution. The human Alg14 protein structure was downloaded from the SWISS-MODEL repository (ID: Q96F25). This model was also evaluated using ProSA and PROCHECK tools, which assessed its overall quality as good . The resulting dimer structure was also assessed using the same tools  indicating that the dimer model has good quality; however slightly lower than the quality of Alg13 and Alg14 models separately.To examine the behavior of the Alg13-Alg14 dimer, we needed to create another homology model, this time of the dimer. We employed the Modeller 9v19 to create a homology model based on two templates of the MurG protein complexed with the substrate—Although the bacterial MurG protein consists of two domains forming a monomer, the human Alg13-Alg14 dimer structurally resembles it. The human Alg14 overlaps with the N-terminal domain, while the Alg13 overlaps with the C-terminal domain. However, the sequence similarity between those proteins is relatively low (below 28% identity). The N-terminal domain of MurG as well as the human Alg14 model, consists of six parallel β-strands surrounded by five α-helices in the case of MurG and seven in the case of the human Alg14 structure. The C-terminal domain of MurG also consists of six parallel β-strands surrounded by seven α-helices and an additional α-helix which interacts with the β-sheet of the N-terminal domain. As previously shown , in the To examine the behavior of the system, we analyzed the RMSD of the two domains separately as well as for the whole complex. We also divided the RMSF plot in order to show each domain separately . In geneOur three reported female patients were diagnosed with ALG13-CDG caused by a single-point mutation c.320A>G, resulting in an Asn107Ser variant of the Alg13 protein. It is one of the most frequently occurring pathogenic variants, but data from the IEF results are still limited. Thus, the two aims of our analyses were: (i) to present the differences in transferrin profiles noted in our patients with ALG13-CDG caused by c.320A>G, who presented with different clinical courses and (ii) to discuss the possible disease mechanism based on protein modeling. The patients’ phenotypic manifestation of the disease varied in regards to epilepsy, the severity of neurodevelopmental delay, and anthropometric parameters. However, as there are no straightforward genotype-phenotype correlations in ALG13-CDG, that was not surprising. Here, we would like to focus on the IEF patterns, which we found to be the most interesting of our results.The first description of IEF of serum transferrin was presented in the ALG13-CDG patient described by Smith-Packard et al. . It showTransferrin IEF is one of the three methods (together with CE and HPLC) currently used worldwide for CDG screening , according to the Congenital Disorders of Glycosylation Final Report 2020 . There aALG13 gene results in the Alg13 Asn107Ser variant. The bioinformatic analysis of the Asn107Ser mutation suggested the deleterious character of the amino acid replacement, according to seven out of eight tools of the PredictSNP server  it can also be lethal. Here, a single-point mutation c.320A>G of the human rver see . On the rver see . The mutALG13 has two isoforms, long ALG13-is1 and short ALG13-is2, which share the N-terminal region (125 amino acids) that hosts the catalytic domain, and the isoforms differ substantially in the C-terminal region. The C-terminal region of the long ALG13-is1 was shown to have ubiquitinase activity, while the C-terminal region of the short ALG13-is2 is thought to be important in shuttling the Alg13 protein to the ER [ALG13-is2 isoform was analyzed by Gao et al., who showed that the C-terminal region of the Alg13 protein is crucial for the interaction with the Alg14 protein [We used a homology model built by the I-TASSER webserver based on several available templates, such as the structures of yeast Alg13. Human o the ER . Moreove protein . Since t protein , predictIn our study, we used three models obtained by different structure prediction methods: hAlg13_hm was predicted by I-TASSER, Alg14 model by SWISS-MODEL, and the dimer model by Modeller. All those tools are template-based, so the accuracy of the predicted model relies strongly on the quality and sequence similarity between the target and template models. To assess the quality of these models we used two different approaches which indicated good quality of the models. It is worth noting that the dimeric structure of the Alg13-Alg14 complex for modeling of which we used other homology models was less accurate. However, as shown in Mycobacterium tuberculosis MurG inhibitors. They showed that during the molecular dynamics, simulations of the apo structure of MurG, the RMSD values fluctuated near 4–6 Å, which indicated that our Alg13 model is relatively stable.Both the Alg13 and Alg14 proteins consist of long loops that connect the secondary structure elements with each other. Such long loops may affect the RMSD and RMSF plots, which we used for the description and evaluation of the analyzed models, since they introduce flexibility. It must be kept in mind that a protein structure requires some degree of flexibility, which allows it to change its conformation in order to interact with substrates, products, and/or other proteins. Therefore, we compared the RMSD plots with those presented by Saxena et al. , who preIn our study, we ran five MD simulations of the apo structure of human Alg13 protein as well as it complex with the UDP-GlcNAc substrate, for both native Alg13 and the Asn107Ser variant. The homologous C-terminal region of MurG, which is called the α/β/α motif, comprises a large part of the donor binding site . It is wE. coli MurG (PDB ID: 1nlm) and P. aeruginosa MurG (PDB ID: 3s2u). Gao et al., who showed that the Alg13 and Alg14 proteins are homologous to the bacterial MurG , also cto Alg13 . They reP. aeruginosa MurG with bound UDP-GlcNAc (PDB ID: 3s2u) and concluded that both Asn107 and Ala81 residues are located in a region that is involved in substrate binding. Therefore, we suspect that despite the fact that the results of the MD simulations of both dimers suggested that the binding of the substrate is similar, the Asn107Ser variant of the human Alg13 may cause issues related to the transport of the substrate.The results of the Alg13-Alg14 dimer simulations showed a different tendency regarding the movement of the Cα atom of residue 107 of human Alg13. When we simulated only the Alg13 protein, for both the apo structure and the complex with the substrate, we observed a higher flexibility of the Asn107Ser variant in the region hosting the mutation. However, during simulations of the dimer structure, the flexibility of both dimer structures was similar. The substrate, during simulations of both dimers, comprising the native and Asn107Ser variant of Alg13, remained in the binding pocket. Therefore, we speculate that the overall Alg13-Alg14 complex is functional, and that the Asn107Ser variant disturbs the substrate transport from Alg13 with bound substrate to the Alg13-Alg14 heterodimer. Increased flexibility of the native Asn107 residue relative to the simulations of the monomeric Alg13 may suggest its role during the catalytic reaction. Our results also confirmed the findings of Ng et al. , who ideALG13 gene results in the Asn107Ser variant of the Alg13 protein. According to the bioinformatic analyses, the mutation itself is not deleterious; however, it causes a multisystemic metabolic disorder, called ALG13-CDG, in the patients with the c.320A>G mutation.A single-point mutation c.320A>G in the Analysis of five repetitions of MD simulations of the homology model of the human Alg13, both as an apo structure or in complex with the UDP-GlcNAc substrate suggests that the Asn107Ser variant alters flexibility of the loop hosting this mutation. Such influence was not observed for the Alg13-Alg14 dimer. The observations from the MD simulations, as well as data from a thorough literature search allowed us to speculate about a plausible mechanism for the role of the Asn107Ser variant of the Alg13 protein during N-glycosylation. While Alg14 is membrane-bound, Alg13 acts as a transporter, it binds the substrate and moves it to Alg14. In the presence of the substrate, Alg13 and Alg14 form a heterodimer in which the second step of the N-glycosylation occurs. Our results suggest that in the case of the Asn107Ser variant of the human Alg13 protein, the Alg13-Alg14 complex remains functional, but the process of the substrate binding to Alg13 and its further transport might be disturbed, thus disturbing the N-glycosylation process.We also showed the IEF results of three female patients diagnosed with ALG13-CDG caused by c.320A>G. Since the ALG13-CDG caused by c.320A>G is very rare, we would like to report on further female patients with different spectra of clinical severity, with abnormal patterns of serum transferrin.


Here, we discuss the presence and roles of heterogeneity in the development of uveal melanoma. Both genetic and cellular heterogeneity are considered, as their presence became undeniable due to single cell approaches that have recently been used in uveal melanoma analysis. However, the presence of precursor clones and immune infiltrate in uveal melanoma have been described as being part of the tumour already decades ago. Since uveal melanoma grow in the corpus vitreous, they present a unique tumour model because every cell present in the tumour tissue is actually part of the tumour and possibly plays a role. For an effective treatment of uveal melanoma metastasis, it should be clear whether precursor clones and normal cells play an active role in progression and metastasis. We propagate analysis of bulk tissue that allows analysis of tumour heterogeneity in a clinical setting. The study of cancer dates back as far as centuries B.C. but it was not until the seventeenth century A.D. that research was properly documented and uncontrolled growth of body cells was described . Over thTo understand the dynamics of cancer progression, it is important to realise that its development is based on somatic evolution . Tumour The resulting diversity, referred to as heterogeneity, is thus present in the very early stages of cancer development and may progress in late progression stages and metastasis , 9. SincDepending on growth advantage of the advanced clone, precursor clones may remain visible. This co-occurrence of precursor clones will allow for the analysis of tumour evolution. In cutaneous melanoma (CM) this has been elegantly demonstrated by dissection and analysis of mixed tumours that contained visible remainders of precursor lesions . HoweverGNAQ/11 Q209L/P and BRAF V600E respectively, are already present in otherwise healthy nevi and are generally not capable of developing into cancer without additional mutations [Uveal melanoma (UM) is an intraocular neoplasm originating from melanocytes in uvea, which comprises the iris, ciliary body and choroid Fig. . UM is autations –19. The utations . These mutations  Fig. 3)GNAQ/11 Qutations . After sUveal melanoma (UM) provides the ideal model to study heterogeneity since it grows in the corpus vitreous, providing a unique pure cancer-cell environment, whereas in other cancers the interference of tissue that is not related to the tumour is common. UM have a slow growth rate with doubling times ranging from 128 to 292 days depending on cell composition . This maCDKN2A gene are candidates for the next step in melanoma development [BAP1, SF3B1 and EIF1AX, have been found in UM but none are as common as the presence of GNAQ/11 [The development of cancer is a multi-step process which has been described in numerous tumour types, and melanoma development should be viewed in a similar fashion , 29. Simelopment –34. For  GNAQ/11 –44.This shows that there are multiple second hits possible, allowing the tumour to divert into different directions and become  heterogeneous with distinct clinical and pathological behaviour. In contrast, intratumorally UM are considered to be homogeneous in a sense that every tumour has its own set of specific mutations , 46. To However, the possibility that UM development can follow different paths, even within one tumour, that lead to successful tumour formation makes the presence of heterogeneity within a tumour likely. This intratumour heterogeneity can be viewed in different ways. If we assume the existence of cancer stem cells (CSC), this pool of cells hierarchically governs the fate of the tumour. Typically, this would mean a high turnover of clones that all share a large portion of genetic aberrations with their parental CSCs . This waAlternatively, recent research in UM indicated a more linear evolution that still results in different subpopulations that represent different progression steps within one UM , 52. SepGiven its slow growth rate, progenitor clones are less likely to get completely overgrown by descendant clones. By means of next generation sequencing of spatially separated biopsies, it has already been possible to detect several sub-populations within one tumour . With a BAP1, SF3B1 and EIF1AX [Next to recurrent mutations, other common anomalies were found in the form of altered chromosomal copy numbers. In UM, especially chromosome 1, 3, 6, 8 and 16 are affected and are just as common as mutations like d EIF1AX –63. Chrod EIF1AX . The mosd EIF1AX –67. Earld EIF1AX –71. Usind EIF1AX . Indeed,d EIF1AX . Given tBesides scientific implications, this concept also has possible clinical consequences. Often, correlations between genetic aberrations and survival are made to demonstrate the clinical relevance of molecular biology of UM , 73–75. GNAQ/11) are present in all tumour cells of most UM, this is considered to be the primary event. As opposed to next generation sequencing, absolute quantification with digital PCR allows to detect small differences in clone fractions and is therefore able to differentiate between early events [Absolute quantification of clones also offers opportunities to quantify precursor and descendant clones . This coy events .Though intuitively one may assume that the most progressed tumour cells will successfully disseminate, metastases may be formed at various stages of tumour evolution. Theoretically, multiple metastases may present with a different genetic set up that require different treatment approaches.Recently, three studies explored the genetic characteristics of matched primary and metastatic UM , 76, 77.UM grow into the acellular corpus vitreous and are therefore considered to have a high tumour purity . However, next to the fraction of cells in a tumour that have genetic aberrations, there is also a part that does not contain genetic defects, often referred to as the tumour microenvironment Fig. 5)..5).Fig.This portion of non-malignant cells can strongly differ between UM , 80. TheIn the healthy state, the eye is considered to be an immune privileged organ, meaning that the blood retinal barrier (BRB), similar to the blood brain barrier (BBB), acts as a physical barrier for both pathogens and immune cells –104. ThiAs a result, lymphocytes have access to the choroid, and they do appear in UM. This still suggests that an active influx of T-cells is possibly triggered by tumour growth. The resulting cellular heterogeneity has implications for comprehending UM dynamics that lead to tumour progression and ultimately metastasis. Unfortunately, it is not known what the cause and the resulting effect is, which would be helpful in clarifying what role the immune system plays in UM development. Can we measure the immune infiltrate and thereby predict tumour behaviour for diagnostic, prognostic and eventually treatment purposes? Historically, the presence of lymphocytes and macrophages was shown histologically , 108. MoDetailed knowledge on the immune microenvironment may reveal opportunities in uveal melanoma treatment. The therapies that have been successful in cutaneous melanoma rarely are effective in uveal melanoma , 76. TheIn UM metastasis, the immune invasion is not as prominent as in the primary UM and often limited to the margins of the tumour . This quIt is impossible to speak about a standard tumour with standard characteristics. Just as our bodies differ from one another, determined by our genetic code, every tumour has its own identity. Although uveal melanomas could be considered genetically homogeneous based on their shared Gαq signalling mutations, occurring in almost all uveal melanomas, subsequent genetic alterations driving tumour progression cause genetic differences between tumour sub-clones.A tumour mass, especially a primary uveal melanoma, should not be seen as a homogenous clump of cells. Although some alterations may be shared between all cancer cells, others are only present in distinct clones and thus a subset of the cells. However, the clinical significance of intratumour heterogeneity in UM has not yet been fully revealed. But it is clear that different clones can metastasise from a heterogeneous tumour, and that this may require adjustments in the treatment of metastatic disease . MoreoveWhen different clones coexist within one tumour, the spatial distribution of the clones may influence the detection of their presence. When a certain clone contains prognostic or therapeutically relevant genetic information, being able (or unable) to identify its presence has immediate clinical consequences. On the other hand, the presence of non-cancer cells should always be taken into account. Apart from its biological relevance, its influence on the detection of genetic alterations is inevitable as it ‘contaminates’ the tumour with a fully healthy genome. A major challenge comes in the exact differentiation of copy numbers. Whereas mutations can be identified as newly acquired changes, never present in any other cell, copy number alterations may present as very limited imbalances .BAP1 and RASSF1A, localised on chromosome 3, accompanied by activation of the MYC oncogene on chromosome 8q may follow this loss of chromosome 3 and could even be the cause of poor survival [By the analysis of different progression stages of UM, it was shown that metastases may actually be derived from precursor clones and not necessarily from the last progression step of the primary UM. This shows again that UM not only grow slowly but also progress slowly. Years after treatment of the primary UM, liver metastases may arise that were not present at the time of treatment. To consider that spreading by a precursor clone would then already have occurred, puts progression and metastatic spreading of UM in a whole new perspective. Detailed knowledge on the lesions present in the primary UM and the UM metastasis and the order in which they occurred is of importance for the molecular evolution and the possibilities for treatment. Eventually this should indicate possibilities to prevent metastases. A full understanding of all the steps in metastasis will eventually allow the development of modalities to inhibit the correlated pathways and prevent metastasis. Monosomy 3 for example defines tumours with a poor prognosis. Inactivation of the tumour suppressor genes survival . Recent survival , 117. Mosurvival . Unravel


Dietary protamine can ameliorate hyperlipidemia; however, the protamine-derived active peptide and its hypolipidemic mechanism of action are unclear. Here, we report the discovery of a novel anti-obesity and hypocholesterolemic peptide, RPR (Arg-Pro-Arg), derived from protamine in mice fed a high-fat diet for 50 days. Serum cholesterol levels were significantly lower in the protamine and RPR groups than in the control group. White adipose tissue weight was significantly decreased in the protamine and RPR groups. The fecal excretion of cholesterol and bile acid was significantly higher in the protamine and RPR groups than in the control group. We also observed a significant decrease in the expression of hepatic SCD1, SREBP1, and adipocyte FAS mRNA, and significantly increased expression of hepatic PPARα and adipocyte PPARγ1 mRNA in the protamine group. These findings demonstrate that the anti-obesity effects of protamine are linked to the upregulation of adipocyte PPARγ1 and hepatic PPARα and the downregulation of hepatic SCD1 via SREBP1 and adipocyte FAS. RPR derived from protamine has a crucial role in the anti-obesity action of protamine by evaluating the effective dose of adipose tissue weight loss. Endocrine disorders, environmental factors and genetic susceptibility cause obesity, which is a severe metabolic disease. It is typically related to other metabolic diseases such as cardiovascular disease, type 2 diabetes, hypertension, and cancer. It is characterized by an abnormal fat-accumulation in the adipose tissue that results from excessive calorie intake derived from an imbalance between energy intake and energy consumption ,2,3,4. NProtamine is a basic protein obtained from fish  generally and consists of heterogeneous proteins which have an average molecular weight of 4500 Da. Approximately 70% of the amino acid composition of protamine is arginine . Jobgen Thus, we aimed to identify the active peptide derived from protamine that was related to both anti-obesity and hypocholesterolemic actions induced by protamine. Based on our research, we hypothesized that RPR derived from protamine is an active peptide related to both anti-obesity and hypocholesterolemic actions induced by protamine. To test this hypothesis, we attempted to identify novel anti-obesity and hypocholesterolemic peptides derived from protamine in animal studies. We also investigated the effects of the novel anti-obesity and hypocholesterolemic peptide, Arg-Pro-Arg (RPR) derived from protamine, in mice fed a HFD.https://web.expasy.org/peptide_cutter/, accessed on 1 March 2019). Protamine is composed of 33 amino acids (MPRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR), and arginine accounts for approximately 70% of the constituent amino acids [In this research, protamine derived from chum salmon milt (Oncorhynchus keta) was used. Chum salmon-derived protamine was digested using in silico analysis with trypsin by PeptideCutter . Mice were housed individually in standard plastic rodent cages and placed in a room that was kept at 22 ± 2.0 °C; there was a 12-h cycle of light (8:00–20:00) and dark, and food and water were freely available. All mice were fed a commercial non-purified diet  and tap water ad libitum for 3 or 5 days before their division into three weight-matched groups. Subsequently, each group was given free access to drinking water and fed the experimental diets for 50 days.There are three experimental groups as shown in The composition of the experimental diets (HFD) was based on the formula recommended by the American Institute of Nutrition (AIN-93G)  and contLipid concentrations in serum, liver, and feces were measured using commercially available kits as follows: Triglyceride E-test Wako  used for serum, liver, and fecal triglyceride, Cholesterol E-test Wako  used for serum, liver, and fecal cholesterol, and with HDL-Cholesterol E-test Wako  used for serum HDL-cholesterol, Phospholipid C-test Wako  used for liver, and fecal phospholipid, and TBA-test Wako  used for fecal bile acid. Liver and fecal lipids were extracted by the method of Folch et al. , and totTM Real-time PCR system (Applied Biosystems) and SYBR® Premix Ex Taq , according to the manufacturer’s protocol. The amplification protocol was as follows; the temperature profile for the reaction was an initial denaturation stage of 95 °C at 30 s, then a two-step program was developed for 40 cycles including denaturation at 95 °C for 5 s, an annealing step and extension step at 60 °C for 30 s, respectively. The following primers were used in the assay based on the NCBI databases  was reverse-transcribed to cDNA using a High-Capacity cDNA Archive Kit . Real-time PCR was performed using a StepOnePlusatabases . Relativp < 0.05, ** p < 0.01. The statistical analysis was performed by JSTAT ver 8.2.Values are expressed as means  ±  SEM. The statistically significant differences were evaluated using one-way ANOVA and Dunnett’s tests . DiffereProtamine was digested with trypsin using PeptideCutter. Protamine tryptic hydrolysate consists of MPR, RRR, SSSRPVR, RRRRPR, VSR, RRRRR, GGR, and RRR by PeptideCutter (protamine is not hydrolyzed by pepsin) in silico. Among them, RRRRPR is hydrolyzed one by one of arginine from the N-terminus by trypsin  and finaWe examined body weight gain, food intake, and liver weight of each mouse in the experiment. There were no significant differences between the control group and protamine or RPR groups regarding growth parameters, namely, initial body weight, food intake, and liver weight. Body weight gain in the protamine group was significantly lower than that in the control group a–c. TherTo investigate whether RPR derived from protamine or protamine induced anti-obesity action in mice fed an HFD diet, various WAT weights were measured. The perirenal or subcutaneous WAT weight was significantly lower in the protamine and the RPR groups than in the control group, and epididymal WAT weight decreased in the RPR group compared to the control group. Total WAT weights decreased significantly in the protamine and RPR groups compared to the control group a,b, suggAs shown in We evaluated the effect of dietary RPR or protamine on fecal lipid excretion in mice. Fecal weights were not significantly different among the groups a. The feWe determined the mRNA expression levels of lipid metabolism-related genes in the liver to evaluate the effects of protamine and RPR using real-time PCR . HepaticWe determined the mRNA expression levels of lipid metabolism-related genes in epididymal WAT to evaluate the effect of protamine and RPR using real-time PCR . PPARγ1 This is the first research to identify RPR (Arg-Pro-Arg) as a novel anti-obesity and hypocholesterolemic tripeptide derived from protamine in mice. The degree of anti-obesity action of RPR treatment was almost identical to that of the protamine treatment, as measured by the WAT weight . Thus, RDecreased circulating total cholesterol and LDL cholesterol concentrations are associated with a reduction in the risk of cardiovascular disease ; therefode novo lipogenesis genes, for example, FAS [SCD1-KO mice showed the reduction of fatty acid and triglyceride synthesis by the high-carbohydrate feeding [SCD1 ablation significantly decreased SREBP1 mRNA level, which was consistent with the observation that SCD1-KO mice showed a reduced expression and maturation of SREBP1, which was specifically rescued by oleate treatment. Oleate can modify the SREBP1 protein and enhance its nuclear translocation, which increases the expression of lipogenic genes [SCD1-KO mice [Hepatic SCD1 or SREBP1 mRNA levels were significantly decreased in the protamine group compared to those in the control group . Some stple, FAS  or SCD1 ple, FAS . A previ feeding . SCD1 abic genes . Thus, t-KO mice . To confThe study of Lodhi et al. has shown that the specific deletion of FAS in adipose tissue increases brown fat-like adipocytes in WAT and increases energy expenditure, which ameliorates diet-induced obesity . Thus, aPepT1KO mice, the cholesterol-lowering effect of FP was mediated through PepT1 in mice [The molecular mechanism related to the suppression of SCD1 via SREBP1 or the activation of adipocyte PPARγ1 and hepatic PPARα by RPR or protamine is unclear. Two relevant mechanisms have already been discovered. Lactostatin (IIAEK), an in vivo active novel hypocholesterolemic pentapeptide, which mediates the calcium-channel-related mitogen-activated protein kinase (MAPK) signaling pathway for cholesterol degradation in HepG2 cells , and the in mice . These o in mice . As RPR  in mice , RPR mayTaken together, this study shows that RPR derived from protamine is a new biological agent for the creation of functional foods and medicines that are expected to prevent and improve obesity and hypercholesterolemia.In summary, our results suggest that RPR derived from protamine or protamine supplementation ameliorates hypercholesterolemia and obesity in HFD mice . RPR or


Perioperative hypothermia is a common occurrence, particularly with the elderly and pediatric age groups. Hypothermia is associated with an increased risk of perioperative complications. One method of preventing hypothermia is warming the infused fluids given during surgery. The enFlow™ intravenous fluid warmer has recently been reintroduced with a parylene coating on its heating blocks. In this paper, we evaluated the impact of the parylene coating on the new enFlow’s fluid warming capacity.2O was infused into each heating cartridge at flow rates of 2, 10, 50, 150, and 200 ml/min. The infused fluid temperature was set at 4 °C, 20 °C, and 37 °C. Output temperature was recorded at each level. Data for analysis was derived from 18 runs at each flow rate (six cartridges at three temperatures).Six coated and six uncoated enFlow cartridges were used. A solution of 10% propylene glycol and 90% distilled HThe parylene coated fluid warming cartridge delivered very stable output of 40 °C temperatures at flow rates of 2, 10, and 50 ml/min regardless of the temperature of the infusate. At higher flow rates, the cartridges were not able to achieve the target temperature with the colder fluid. Both cartridges performed with similar efficacy across all flow rates at all temperatures.At low flow rates, the parylene coated enFlow cartridges was comparable to the original uncoated cartridges. At higher flow rates, the coated and uncoated cartridges were not able to achieve the target temperature. The parylene coating on the aluminum heating blocks of the new enFlow intravenous fluid warmer does not negatively affect its performance compared to the uncoated model. Between 25 and 90% of all patients who undergo elective surgery experience hypothermia, defined as a body temperature below 36 °C . AnestheHypothermia is associated with an increased risk of complications, some of which can be severe , 5. SeveNumerous methods have been devised to prevent inadvertent hypothermia , 11. SurCurrent guidelines recommend incorporating a method of warming infused fluids during surgery , 15, 17.Kim et al. studied the effect of higher flow rates of fluid on output temperature over time . In theiIntravenous fluid warmers based on aluminum heating blocks have been troubled with aluminum leaching into the fluid, leading to concerns about aluminum toxicity –27. The Heat transfer from the aluminum block to the passing fluid takes place through forced convection. The equation is expressed as Newton’s Law of Cooling:Q is the heat transfer to the liquid in Watts.c is the convective heat transfer coefficient for the process and is a function of both the liquid velocity and liquid properties for a given fluid.hA is the area of plate in contact with the liquid in the cartridge.and dT is the temperature difference between the plate and the fluid.2.K [The heat transfer coefficient in forced convection for aluminum falls between (59-64)w/m2.K . This co2O. A 10 L capacity chiller tank  was filled with the 10% PG solution, and the solution was maintained in one of three different temperature ranges: cold (4 °C), normal (around 20 °C), and warm (more than 35 °C). The conditioned samples were then maintained in the temperature range using the chiller tank. Five different flow rates were tested in this study: 2 ml/min, 10 ml/min, 50 ml/min, 150 ml/min, and 200 ml/min. A pump  was used to generate the different flow rates. The peristaltic pump was calibrated each time the tubing or cartridge was changed using a volume per unit of time procedure, according to the pump manual instructions. A standard non-insulated extension tubing set  was connected at the fluid warmer outlet. Measurements were performed in a laboratory setting at 21-24 °C room temperature. The fluid temperature was measured at the input (Tin) and the output (Tout) of the enFlow cartridge  were recorded by a laptop computer with data acquisition terminal using Tera Term .In this study, we evaluated the efficacy of two fluid warmers (parylene-coated and uncoated enFlow cartridges) under several conditions. A solution with thermal properties equivalent to 5% dextrose was produced using 10% propylene glycol (PG) and 90% distilled Hin and Tout were obtained (six cartridges at three temperatures each) for each of five flow rates and used for the statistical analysis .The parylene-coated enFlow cartridge begins delivering target temperatures within seconds of activation of the heating unit. Starting at 2 min after the fluid infusion was begun to allow for equilibration, the input and output fluid temperatures were recorded and averaged over 15 s. The measurements were repeated for six different coated and uncoated enFlow cartridges to evaluate inter-device variability. A total of 18 data points for TStatistical analysis and graphical presentation were performed using GraphPadPrism 9.2.0 .out is plotted with error bars representing the standard error of the mean. The parylene-coated fluid warming cartridge delivered target output temperatures at flow rates of 2, 10, and 50 ml/min regardless of the temperature of the infusate. At the lowest flow rates (2 ml/min and 10 ml/min), both enFlow cartridges achieved their target set temperature for all fluid temperatures.Figure At the higher flow rates (150 and 200 ml/min), the heating performance was also similar between both warmers. Output temperatures were clearly impacted by the input temperatures at 150 and 200 ml/min. With an input temperature of 4 °C, the parylene-coated enFlow increased the temperature of the fluid by 23.5 °C at 150 ml/min and by 18 °C at 200 ml/min. With an input temperature of 20 °C, the parylene-coated enFlow increased the temperature of the fluid by 19.7 °C (to 39.8 °C) at 150 ml/min and by 17.3 °C (to 36.7 °C) at 200 ml/min. With an input temperature of 37 °C, the parylene-coated enFlow cartridge increased the temperature to 40 °C at all flow rates.We compared the warming capacity of two different models of the enFlow intravenous fluid warming cartridge. We tested different flow rates and varying fluid input temperatures using the uncoated and coated fluid warming cartridges. At lower flow rates, both types of enFlow cartridge achieved warming of fluid to the target temperature, regardless of the input temperature. Direct comparisons are difficult due to differences in experimental setups. At higher flow rates, the results showed similar heating performance between the two devices. According to these results, the parylene coating does not appear to negatively impact the heating capacity of the cartridge.out between the devices, it is unclear whether these differences are in fact clinically significant. Although 1-2 °C seems trivial at first examination, human temperature is very tightly controlled by the thermoregulatory system. Decreases of only 0.2 °C are associated with homeostatic processes designed to maintain temperature, and a drop in core temperature of 1 °C triggers shivering [While there were subtle differences in Thivering , 30. Whiin, the differences between the set point and Tout increased. Resultant temperatures in the uncoated cartridge at 2 ml.min−1 fell off by approximately 2 °C, a drop not seen at 10 ml*min−1. As we see this decreased temperature only at very slow flow rates, we believe that this decrease in temperature is caused by the cooling of the fluid as it courses from the output of the enFlow device to the temperature sensor. The heated fluids are flowing more slowly through the tube, allowing greater time for heat loss before reaching the sensor [As seen in Fig. e sensor .Parylene is a polymer that is applied to electrical circuits and medical devices. It forms a pinhole-free layer at 14 Å. It forms a chemical and moisture barrier at only 10 Å thick, effectively insulating the aluminum heating plates from the infusate . The parParylene is typically a thermal barrier as well as a chemical and electrical barrier. Parylene has a thermal conductivity rated at 0.082 watts/meters·Kelvin, while water and glass are rated at 1.13 W/m·K and silicone is 163 W/m·K . DespiteThere are several studies evaluating the performance of fluid warmers in the literature , 35–37. This study has the strength that it evaluated for the first time the impact of a parylene coating on the heating performance of fluid warmers and tested the system in flow rates up to 200 ml/min. Further for the first time, testing was repeated in six enFlow cartridges to confirm the repeatability of the results. Given the current concerns related to aluminum toxicity , 38, thiNotably, we were not able to identify any previous studies of fluid warmer performance at such high flow rates. Infusions at 150 or 200 ml/min are not unusual in the clinical setting and yet the performance of fluid warmers at these infusion rates has not been described. High infusion rates are often used for trauma patients requiring rapid expansion of blood volume due to hemorrhage, for example, or for patients undergoing open abdominal surgery who may lose fluid quickly through evaporation and third-spacing. The coated and uncoated enFlow system was able to warm the infusate towards physiologic temperature even at these high rates.Our study has some limitations. We only studied the impact of parylene coating on one fluid type, and the efficacy of the device may be different using fluids with higher or lower viscosity such as saline, whole blood, fresh frozen plasma, or infusions high in lipids. Waldmann et al  evaluateThe parylene coated enFlow cartridges warm the infused fluids at a level and rate that are consistent with those of the uncoated enFlow cartridge. The parylene coating on the aluminum heating blocks of the new enFlow intravenous fluid warmer does not affect its performance compared to its uncoated model. Parylene coating overcomes the recently described aluminum leaching, without reducing the heating capacity of the enFlow system.


In the early 1900s, German chemist, Paul Ehrlich commenced developing drugs to treat infectious diseases and coined the term ‘chemotherapy’ defining it as the use of chemicals to treat disease. He developed the first alkylating agents, to treat cancer.a magic bullet with a single drug able to kill tumour cells,a magic bomb combining surgery, radiotherapy and chemotherapy to achieve higher levels of anti‐tumour efficacy,monoclonal antibodies to receptors over‐expressed on tumour cell surface to reduce toxic side effects and achieve tumour‐targeted therapy,synthetic nanoparticles and polymers to package drugs and deliver them to tumour cells to reduce toxic side effects and achieve higher anti‐tumour efficacy,molecularly targeted drugs that targeted specific oncogenic proteins to avoid killing normal cells,checkpoint inhibitor immunotherapy to reactivate the patient’s own immune system,Chimeric antigen receptors to give T cells the ability to target a tumour‐specific protein and T cell activating function in a single receptor.During the past 100 years, the major ‘cancer cure’ concepts evolved from;While some spectacular cures were observed with each of these approaches, most patients experienced tumour relapse and eventually succumbed to the disease. These advances provided an incremental advance in the treatment of cancer and almost all of them were associated with moderate to severe toxicity. Throughout this time, there was a major effort to discover antigens that were tumour‐specific with a hope that tumour‐targeted therapies could be developed with minimal toxicity to normal tissues. This effort has yet to bear fruit.et al., Most cancers are diagnosed when patients are at Stage III or IV. It is well known that tumour heterogeneity at this stage is extensive and most tumour cells have already elaborated multiple drug resistance and immune‐suppression mechanisms. Cell proliferation assays on tumour cells isolated from biopsies show near complete resistance to most available chemotherapeutic drugs  could alleviate the symptoms of asthma or chronic obstructive pulmonary disease, penicillin and other antibiotics could cure bacterial infections. The same principle has been applied to cancer therapy for example, when it was discovered that cytotoxic T cells were being suppressed by PD1 ligands secreted by tumour cells, it was thought that anti‐PD1 inhibitor monoclonal antibody would reactivate CD8+ T cells and would effect a cure. While anti‐PD1 inhibitors do help in some cancers, the disease is far too complex with thousands of molecular events in action simultaneously to allow tumours to escape the immune system and aggressively overcome host defences  PNU‐159682, a metabolite of the anthracycline nemorubicin, a highly potent DNA topoisomerase I inhibitor which is over 2000‐fold more toxic than conventional drug doxorubicin, (ii) Duocarmycin which is a DNA minor groove‐binding alkylating agent, (iii) Maytansine, a benzoansamacrolide, a highly potent microtubule‐targeted compound that induces mitotic arrest and kills tumour cells at sub‐nanomolar concentrations etc. Unfortunately, these drugs cannot be administered in patients as free chemotherapy since they are too toxic and would kill a person due to rapid and widespread killing of normal cells. These drugs are being developed as antibody‐drug conjugates but even then, they are seriously toxic in patients.If it were possible to safely administer these drugs into cancer patients so that the drug is specifically taken up inside cancer cells and not normal cells, then it should be possible to kill even the most drug‐resistant cancer cells. Bacterial minicells which are anucleate nanoparticles produced as a result of inactivating the genes that control normal bacterial cell division   they are retained in the normal blood circulation since the gaps between endothelial cells lining the blood vessels is less than 2 nm. However, it is known that cancer cells require access to blood vessels for growth and metastasis and hence they over‐express pro‐angiogenic factors which leads to the development of disorganized blood vessel networks that are fundamentally different from normal vasculature. Tumour vasculature is typified by aberrant structural dynamics and vessels that are immature and hyperpermeable Siemann, . The fenThe EDVs being 400 nm rapidly fall out of these fenestrations and enter into the tumour microenvironment and since they carry the bispecific antibody on the EDV surface, the anti‐EGFR component binds to EGFR on the tumour cell surface. This provokes macropinocytosis and the EDVs are taken into the early endosomes, followed by lysosomes and broken down in these organelles releasing the drug PNU‐159682. The drug enters into the tumour cell cytoplasm and the nucleus and intercalates with the chromosomal DNA resulting in tumour cell apoptosis. In the event that a tumour type does not express EGFR for example liver cancer, which expresses asialoglycoprotein, then the bispecific antibody can be changed to anti‐asialoglycoprotein while the anti‐O‐polysaccharide component remains constant. Similarly, HER‐2 positive breast cancers can be targeted via anti‐HER2/anti‐O‐polysaccharide bispecific antibody.et al., et al., This is the first time a super‐cytotoxic drug has been administered into human cancer patients with no toxicity. The double membrane structure of the EDVs prevented leakage of the drug in general circulation, the large size of the EDVs allow it to avoid the normal tissues that are surrounded by normal sealed blood vessels, the tumour‐associated leaky vasculature allows the EDV to enter specifically into the tumour microenvironment, the bispecific antibody targeting of the EDVs allow it to enter specifically into tumour cells and the lysosomal degradation machinery allowed the EDV to be broken down intracellularly and release the drug that could overcome drug resistance and for the first time, kill tumour cells that are highly drug resistant with no toxicity. All 170 cancer patients treated so far were end‐stage palliative care patients who had run out of all treatment options. Highly significant anti‐tumour efficacy has been observed in mesothelioma  such as LPS by professional phagocytes (APCs) being macrophages and dendritic cells (DCs) which are present in the lymph nodes, liver and spleen. Recognition of PAMPS results in the APCs releasing ‘alarm signals’ like ATP  release ‘find‐me’ signals such as low levels of nucleotides ATP and UTP, fractalkine, lysophosphatidyl choline, or sphingosine 1‐phosphate, which attract APCs to the sites of death within the tissue Gregory, . The apoet al., This cascade of events continues to escalate as more APCs are attracted to the new dying tumour cells, engulf them, go to the draining lymph nodes and activate more CD8+ cytotoxic T cells. This cascade then results in tumour antigen‐specific CD8+ effector memory T cells that provide long‐term immunity to the specific cancer (Sagnella The polar sites of septum formation in Gram −ve and Gram +ve bacteria are thought to be vestigial sites of cell division left over from bacterial evolution and it is possible that the minicell may have been the primordial cell during the path of bacterial evolution. The minicell may therefore be considered to be a very early ancestor of bacteria that existed millions to possibly billions of years ago.Today, it re‐emerges to offer a way forward in the intractable problem of cancer treatment. These minicells offer solutions required for treatment of late‐stage cancers with little toxicity, both by killing cancer cells and stimulating a robust anti‐tumour immune response, in effect, allowing the patient’s own immune cells to help in the heavy lifting of getting the patient back on his/her feet. No drug or immunotherapy comes near the minicell as a one‐stop‐shop for cancer treatment and for the next 15 years, the bacterial minicell is likely to radically change how cancer is treated.No funding information provided.The authors have no conflict of interest to declare.


Correction to: Scientific Reports, 10.1038/s41598-020-58717-w, published online 21 February 2020The original version of this Article contained an error in the Acknowledgments section.“This work was supported, in part, by NHLBI R01HL119326 and CDC/NIOSH U01-OH011300 and was also performed at the Preclinical Imaging Laboratory, a shared resource partially supported by the Laura and Isaac Perlmutter Cancer Center Support Grant NIH/NCI 5P30CA016087 and NIBIB Biomedical Technology Resource Center Grant NIH P41 EB017183.”now reads:“This work was supported, in part, by NHLBI R01HL119326, CDC/NIOSH U01-OH011300, the Stony Wold-Herbert Fund and was also performed at the Preclinical Imaging Laboratory, a shared resource partially supported by the Laura and Isaac Perlmutter Cancer Center Support Grant NIH/NCI 5P30CA016087 and NIBIB Biomedical Technology Resource Center Grant NIH P41 EB017183.”The original Article has been corrected.


Postoperative intrapericardial adhesion increases the risk of complications in patients undergoing reoperation. We investigated the effect of a bioabsorbable dextrin hydrogel (DHG) on the formation of intrapericardial adhesions.Intrapericardial adhesion was surgically induced in Japanese white rabbits with DHG treatment (Adh + DHG) or without DHG treatment (Adh). The sham group was not treated with DHG and intrapericardial adhesion was not induced. The extent of intrapericardial adhesion was assessed by adhesion scoring and crystal violet staining of the pericardial cavity. Bromodeoxyuridine (BrdU) uptake assay was performed to assess the proliferative response to the injury in the tissue beneath the intrapericardial adhesion.p < 0.01). The induction of intrapericardial adhesion resulted in a proliferative response mainly in the cardiac tissue just beneath the adhesion. There were 48.6 ± 10.7 cells/0.1 mm2 BrdU-positive cells in the Adh + DHG group and 135.7 ± 23.8 cells/0.1 mm2 BrdU-positive cells in the Adh group on day 28 (p < 0.05).The Adh + DHG group showed looser intrapericardial adhesions compared to the Adh group. The adhesion area of the Adh + DHG group was 4.6 ± 2.2%, whereas that of the Adh group was 32.6 ± 6.4% at the end of the 28-day observation period (These findings indicate that DHG effectively prevented intrapericardial adhesion in this model. Cardiovascular diseases are becoming increasingly prevalent in our aging society. Since the risk factors, including age, are common to various cardiovascular diseases, the chances of the same patient presenting multiple cardiovascular diseases is high . It is eThe sequence of events in postoperative intrapericardial adhesion has been well characterized . The iniOther than surgical technique to minimize the injury and removal of the blood and clots, several approaches have been proposed to prevent intrapericardial adhesion, including pharmacological suppression of inflammation and cell proliferation, and placement of physical barriers . HoweverThe experimental protocol was approved by the Animal Experiment Committee of Kurume University School of Medicine, and animal care and usage were performed in accordance with standard guidelines recommended by the Science Council of Japan. All procedures were performed using standard aseptic techniques, and animals were carefully monitored during recovery from anesthesia. We used male Japanese white rabbits that weighed 2.5–3.0 kg at the time of the experiment.n = 19) and with DHG treatment . Sham operation was performed on 9 animals with the same procedure except the opening of the pericardium and the induction of intrapericardial adhesion. A group of 5 animals without any surgery (Pre-group) was used to observe the tissue without any intervention.To test the effect of DHG, intrapericardial adhesion was induced by surgery. The animals induced for intrapericardial adhesion were divided into two groups: without DHG treatment  were sprayed on the epicardial surface of the anterior wall of the heart and the pericardial surface at a pressure of 0.1 MPa according to the manufacturer’s instructions for clinical use in abdominal surgery , or at the postoperative day 7, 14 and 28 by carbon dioxide inhalation as recommended in the Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan, and the heart was obtained en bloc with surrounding tissue including the sternum and pericardial sac.The intrapericardial adhesion was assessed according to the scoring system previously reported  as folloWe also developed a method for the evaluation of intrapericardial adhesion using crystal violet, a water-soluble dye. At the time of sacrifice, approximately 0.4 mL aqueous solution of 0.4% crystal violet was gently injected into the posterior surface of the pericardial sac without any adhesion until the pericardial cavity was slightly distended Fig. b, and le3 buffer (pH 9.8), and continuously infused using an osmotic pump with a 14-day infusion  that was implanted into the abdominal cavity. The pumps were implanted either at the time of chest surgery or 14 days after the surgery to examine the timing of cell proliferation after the induction of intrapericardial adhesion. The animals with osmotic pump implantation at the time of surgery were sacrificed on day 7 (n = 6 in each group), on day 14 (n = 6 in each group), and on day 28 . The animals with osmotic pump implantation on day 14 were sacrificed on day 28 . We also assessed BrdU uptake in the sham group on days 7, 14, and 28. Animals without any intervention or BrdU infusion (Pre-group) served as a negative control.To evaluate the inflammatory and wound repair response during the formation of intrapericardial adhesion, we assessed cell proliferation based on the uptake of bromodeoxyuridine (BrdU), a thymidine analog. The BrdU solution  was prepared in sterile 0.5 mol/L NaOH/NaHCO2.After evaluation of the intrapericardial adhesion, a 5-mm slice of the whole heart with pericardium at the level of papillary muscles was fixed in 10% neutralized buffered formalin solution. The samples were embedded in paraffin blocks and sectioned (5 μm). Histochemical staining was performed with hematoxylin–eosin (HE) or Masson trichrome (MT) staining. Immunohistochemical staining was performed using anti-BrdU antibody  and nuclear counterstaining with hematoxylin. For the assessment of the tissue response, the anterior ventricular wall including the pericardium beneath the sternum was divided into three regions: the outer, middle, and inner regions. Digital microscopy images were obtained using a BZ-9000 microscope  equipped with a 40X objective lens. The BrdU-positive cell numbers were the average of three digital images with 1360 × 1024 pixels corresponding to 362 × 273 μm from each site, and expressed as the cell numbers in 0.1 mmp < 0.05.Data analysis was performed using GraphPad PRISM 5 . For scoring adhesion, a statistical analysis was performed using Fisher’s exact test. The results are expressed as means ± standard errors. For other assays, comparisons were made using the Kruskal–Wallis test followed by Dunn’s multiple-comparison test. Significance was set at DHG was evident on the epicardial and pericardial surface, just after the surgical procedure to induce intrapericardial adhesion and the application of DHG Fig. a, but waThe hearts of the sham-operated animals without the induction of intrapericardial adhesion showed a smooth outer myocardium. This was covered with a pericardium that contained little cellular components as shown by HE staining Fig. a, b and 2 on days 7, 14, and 28, respectively, when BrdU administration was started on the day of the induction of intrapericardial adhesion. The number of BrdU-positive cells was reduced to 39.0 ± 19.2 cells/0.1 mm2 on day 28 when BrdU was administered only during the last 14 days of the 28-day observation period (Day 28-s in Fig. 2 on days 7, 14, and 28, respectively, compared to Adh group (Fig. BrdU uptake, an indicator of cell proliferative response, was undetectable in the ventricular wall in sham-operated animals Fig. . BrdU-poIn this study, we demonstrated that DHG effectively prevented intrapericardial adhesion in an animal model. DHG-treated animals showed looser intrapericardial adhesion that could be easily dissociated from the heart, as assessed by the adhesion score. The adhesion area, as assessed by crystal violet staining, was also much lower in the DHG-treated animals. Notably, although the adhesion area was comparable between the Adh and Adh + DHG groups on day 7, the adhesion score was lower in the Adh + DHG group. On day 28, both adhesion score and adhesion area were lower in the Adh + DHG group than in the Adh group. Histologically, the Adh + DHG group showed lower cellular infiltration and BrdU-positive cells, indicating that DHG attenuated the injury response.Our results showed that intrapericardial adhesions are associated with the activation of cell proliferation. Although we did not specifically determine the cell types in the current study, previous studies have shown that pericardial mesodermal cells, fibroblasts, and inflammatory cells proliferate during the early phase of intrapericardial adhesion formation –10. The In the Adh + DHG group, although the adhesion area was comparable to that of the Adh group on day 7, both the adhesion score and the count of BrdU-positive cells were lower than those in the Adh group. The adhesion area and score were lower in the Adh + DHG group than in the Adh group on day 28. These findings suggest that a loose adhesion was formed on day 7, which prevented crystal violet staining in the Adh + DHG group, and the adhesion was resolved subsequently, possibly because the injury response was suppressed by DHG, as demonstrated by the lower number of BrdU-positive cells on day 7. While DHG forms a thick coating over the sprayed surface, it is disassembled and absorbed within 3 days in the abdominal cavity , 14. AccSeveral issues need to be addressed before the clinical use of DHG for preventing intrapericardial adhesion. First, the safety of DHG use needs to be tested in the context of cardiac surgery. Although the general safety of DHG is established in surgeries performed in the abdominal cavity, the biological response may be different in the pericardial cavity. As the physical capacity of the pericardial cavity is significantly smaller than that of the abdominal cavity, and due to the risk of causing cardiac tamponade , the volAlthough further research is required before introducing DHG to clinical practice, our results demonstrated that it effectively prevented postoperative intrapericardial adhesion in the animal model. Prevention of intrapericardial adhesion would benefit patients who undergo repeated cardiac surgeries.


Acetes sp) with coarse salt and granulated sugar in a certain ratio. This research was aimed at studying the effect of adding garlic and red chilli pepper powder on the physicochemical, microbiological, and sensory properties of cincalok. Cincalok was made to be three recipes, namely, original cincalok, A, consists of 2 kg of rebon shrimp, 400 g of granulated sugar, and 100 g of coarse salt; B (A ingredients plus 20 g of red chilli pepper powder); and C (A ingredients plus 20 g of garlic powder). Sensory analysis was conducted on recipe A, and the colour was observed by the naked eye on days 0, 2, 4, 6, 8, 13, 18, 23, 28, 33, 43, 60, 90, 120, 150, and 180. According to the highest criterion score on sensory results, the panellists chose day 6 as the best fermentation for recipe A. The colour of recipe A started changing from pink to a light brown colour on the surface on day 28. Therefore, the physicochemical, microbiological, and sensory properties of each recipe were analyzed for 28 days. Overall, the water, ash, and fat content; titratable acid (TA); total volatile base nitrogen (TVBN); and amino acid nitrogen (AAN) showed insignificant differences (p > 0.05) among the recipes during 28 days of the same observation. The crude protein, pH, and free fatty acid (FFA) of recipe C were significantly different (p < 0.05) from recipes A and B. All recipes contained the total count of mesophilic anaerobic bacteria (TMABs) and the lactic acid bacteria (LABs) except Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, and Enterobacteriaceae for all observation times. The highest criterion score for consumer acceptability was awarded for recipe C followed by recipes B and A. The addition of garlic and red chilli pepper powder affected the physicochemical, microbiological, and sensory properties of cincalok.Cincalok, a traditional fermented shrimp, is prepared by mixing rebon shrimps ( Cincalok is a small shrimp fermentation production that is known in South Asia with various names, i.e., cincalok or kecaluk or ronto from Indonesia, cincalok from Malaysia, and balao-balao from the Philippines –4. CincaCapsicum annuum L. var. Longum Sendt) is rich in vitamins, minerals, and capsaicin [Allium sativum L) contains allicin that plays a role in antimicrobial activities [in vitro and vivo [Chilli pepper and garlic are seasonings to develop food aroma, flavour, taste, and appearance. Chilli pepper and garlic are also widely used in fish fermentation and can contribute to increasing the safety of products . Red chiapsaicin . Capsaicapsaicin . Besidesapsaicin . Garlic tivities , 13. Somtivities , 15). Thtivities . The Korand vivo .In this study, the original cincalok (recipe A) was supplemented with red chilli pepper powder (recipe B) and garlic powder (recipe C) and fermented. Physicochemical, microbiological, and sensory properties of cincalok recipes A, B, and C were analyzed. This study was aimed at investigating the effect of adding garlic and red chilli powder in each recipe on the physicochemical, microbiological, and sensory properties of cincalok.Rebon shrimps were bought from a fisherman in Sungai Duri, Bengkayag Regency, West Kalimantan, Indonesia, then directly made cincalok at a cincalok producer in Sungai Duri, Bengkayang, West Kalimantan. Cincalok was prepared using three recipes, namely, A , B , and C . Fresh rebon shrimps  were sorted out from the impurity  then rinsed with seawater. The fresh and cleaned rebon shrimps were carefully mixed with all of the ingredients following the recipes and put in a glass container, covered with a lid, and then incubated at room temperature. Sensory evaluation of each recipe was carried out in Sungai Duri, Bengkayag Regency, West Kalimantan, Indonesia. At the same time, the physicochemical and microbiological analyses were conducted in the Biotechnology Laboratory, Faculty of Mathematics and Natural Sciences, Universitas Tanjungpura, Indonesia.In this study, we divided two steps in cincalok production. In the first step, the sensory and colour were evaluated in recipe A using the naked eye on days 0, 2, 4, 6, 8, 13,18, 23, 28, 33, 43, 60, 90,120, 150, and 180. These results were used to determine the duration of physicochemical, microbiological, and sensory analysis for recipes A, B, and C.Each sample was homogenized using a hand blender before physicochemical analysis.The proximate analysis  in this study was carried out based on the procedures described in the AOAC official method  and IndoTwo g of homogenate was diluted with 18 mL of distilled water. The mixture was measured using a pH-meter digital .The FFA was analyzed using acid-base titration . A 28.2 The TA was determined by acid-base titration according to the AOAC official method 942.15 . One g oThe TVBN was determined using steam distillation described by the Codex Alimentarius Committee (1968) . Twenty-The AAN was determined by formol titration . A two mμL of the suspension sample (dilution 10−1) was inoculated on a specific media and incubated at 30°C for analysis of the total count of mesophilic anaerobic bacteria (TMABs), total count of halotolerant bacteria 10 (THBs 10), THB 17.5, lactic acid bacteria (LABs), fungi, Bacillus cereus, Clostridium perfringens, Enterobacteriaceae, and Staphylococcus aureus. The TMABs, THBs 10, and THB 17.5 were determined using PCA media enriched with 15 g/L, 10 g/L NaCl, and 17.5 g/L of NaCl, respectively [Microbiological analysis was conducted following suspension of A 5 g of sample in 45 mL of sterilized saline buffer (0.9% of NaCl) to obtain a suspension. 50 ectively . Each ba3 and anaerobically incubated was used [For LAB count, MRS media enriched with 1% of CaCOwas used . A clearOxytetracycline Glucose-Yeast Extract Agar (OGYE) media supplemented with NaCl 5%, streptomycin sulfate 0.25 mg/mL and penicillin G 0.25 mg/mL [Determination of fungal total was carried out on 25 mg/mL . The funMannitol Egg Yolk Polymyxin (MYP) supplemented with 5% NaCl and incubated on days 2-3 to determine B. cereus [Bergey's Manual of Determinative Bacteriology [The sample was inoculated on . cereus . The pineriology .S. aureus was determined using Mannitol Salt Agar (MSA) supplemented with 5% NaCl. The yellow colonies with yellow zones were tested for coagulase; then, the positive coagulase test was counted as log CFU of S. aureus.Enterobacteriaceae was evaluated following a procedure described by and Han et al. and Viji et al. using a selective medium, Violet Red Bile Glucose Agar (VRBG), supplemented with NaCl 5% [ NaCl 5% , 27. TheTryptose Sulfite Cycloserin Agar (TSCA) supplemented with NaCl 5% was used to count the total of C. perfringens.The physicochemical analysis of this experiment was conducted using a completely randomized design and repeated three times. The variation among the treatments was analyzed through one-way analysis of variance (ANOVA) using Tukey's test as the post hoc comparison test . Recipe A showed a light pink colour on day 0. The light brown colour on the cincalok surface appeared on the 2833rd day . The broth day can be proposed based on the colour and product appearance. However, the addition of garlic and red chilli pepper powder affected the product appearance of cincalok.In the second step, the researchers further observed the colour and texture for recipes A, B, and C on days 0, 2, 4, 6, 8, 18, and 28. Recipes A, B, and C exhibited different pink colours. Recipe A was slightly brighter pink than recipe B, which showed a dark pink colour caused by the addition of red chilli pepper powder . The addThe commercial cincalok from West Kalimantan is usually ripe after fermentation for 3 to 6 days . Recipesp > 0.05) (p < 0.05) in the same recipe with different incubation times except for the fat content of recipes A, B, and C and the moisture of recipe B (p > 0.05) the proximate compositions.The proximate compositions of recipes A, B, and C were determined  on days 0, 2, 4, 6, 8, 18, and 28 . The pro > 0.05) . The prorecipe B . The addp < 0.05) fermentation time for recipes A and C but not recipe B. The final moisture among the recipes was around 60-61%. It is lower than the commercial cincalok from Indonesia, approximately 67.6% [The moisture on day 0 was around 54-55% for all recipes . The moip < 0.05) toward recipes A and C. Microorganism activities during the fermentation could cause fluctuation in the ash content. For example, LAB-fermented sorghum flour and LAB-fermented maize flour significantly increase (p < 0.05) than naturally fermented sorghum flour [Ash content indicates minerals and inorganic content in food. The ash content of each recipe during the fermentation showed a fluctuation pattern, mainly for recipe C . Besidesum flour .p < 0.05) in each recipe with different fermentation times or with the same fermentation time.The fat content of rebon shrimp as a raw material was low, approximately 0.6-1.9% . The fatLactobacillus plantarum [p > 0.05) during the observation.The crude protein trend tended to decrease for all recipes during the observations . The prolantarum . The cruThe acidity affects food flavour, colour, taste, and preservation, which is an important parameter for assessing product quality . The TA p < 0.05) than that of recipe C. Lower pH of recipes A and B than recipe C might be caused by microorganisms other than LABs.The pH of all the recipes for certain fermentation times significantly decreased from 8 to 5, but there are insignificant differences among recipes for the particular observation time . The recp > 0.05) among recipes A, B, and C but significantly different from each recipe during the fermentation time (The TA showed an insignificant difference (ion time . The addp > 0.05) the TVBN value among the recipes.Protein degradation during fermentation is caused by the activities of proteolytic bacteria and proteolytic enzymes that can be measured from the TVBN and FAN values. The TVBN measures the total amount of ammonia and amines (dimethylamine and trimethylamine). The TVBN value can be used to assess food quality, particularly seafood spoilage. The TVBN also is used to measure the ripeness level of fermentation . The TVBp < 0.05) during the fermentation of each recipe event though insignificant differences among the recipes were observed  during the fermentation period, while the cincalok recipe for the same fermentation time generally showed an insignificant difference (p < 0.05)  . The garB. cereus, C. perfringens, and S. aureus), and Enterobacteriaceae on days 2, 4, 6, 8, 18, and 28. All recipes did not contain moderate halophilic bacteria when they were analyzed using procedures THBs 10 and THBs 17.5, respectively. All of the recipes also did not detect fungi, B. cereus, C. perfringens, S. aureus, and Enterobacteriaceae for all observation times. The commercial cincalok from West Kalimantan was reported by Nofiani and Ardiningsih containing THBs 10, THBs 17.5, fungi, and Enterobacteriaceae [The microbiological profile of products is a fundamental step for assessing the safety and quality of the food products, particularly the ones based on a spontaneous fermentation such as cincalok. The microbiological profile also affects the sensory, textural, and nutritional properties. The microbiological profiles of each recipe were evaluated for the total count of mesophilic anaerobic bacteria (TMABs), total count of halotolerant bacteria 10 (THBs 10), THBs 17.5, lactic acid bacteria (LABs), fungi, some pathogenic bacteria (eriaceae . Lactic p > 0.05) but a significant different (p < 0.05) pH  pH . A low pnd-day fermentation of each recipe, LABs around 4 log CFU/g for recipes A and B were detected, while it was around 3 log CFU/g in recipe C  for the pH, crude protein, and ash but were significantly different (p < 0.05) for recipe C. All recipes showed differences in the microbiological properties such as the TMABs and LABs. The THBs 10, THBs 17.5, fungi, B. cereus, C. perfringens, S. aureus, and Enterobacteriaceae were not detected for all recipes on day 6. However, recipe C (addition of garlic powder) was the most preferred by the consumers then followed with recipes B and C on day 6. The physicochemical, microbiological, and sensory results of cincalok were affected by adding garlic and red chilli pepper powder.All cincalok recipes showed the best maturity on day 6 based on the sensory analysis. On that day, recipes A, B, and C exhibited similar physicochemical properties, notably the TA, TVBN, AAN, FFA, water, and fat content. Recipes A and B showed insignificant differences (


Active fluid droplets surrounded by oil can spontaneously develop circulatory flows. However, the dynamics of the surrounding oil and their influence on the active fluid remain poorly understood. To investigate interactions between the active fluid and the passive oil across their interface, kinesin-driven microtubule-based active fluid droplets were immersed in oil and compressed into a cylinder-like shape. The droplet geometry supported intradroplet circulatory flows, but the circulation was suppressed when the thickness of the oil layer surrounding the droplet decreased. Experiments with tracers and network structure analyses and continuum models based on the dynamics of self-elongating rods demonstrated that the flow transition resulted from flow coupling across the interface between active fluid and oil, with a millimeter–scale coupling length. In addition, two novel millifluidic devices were developed that could trigger and suppress intradroplet circulatory flows in real time: one by changing the thickness of the surrounding oil layer and the other by locally deforming the droplet. This work highlights the role of interfacial dynamics in the active fluid droplet system and shows that circulatory flows within droplets can be affected by millimeter–scale flow coupling across the interface between the active fluid and the oil. Active fluids can self-organize into circulatory flows that are sensitive to confinement shape25. However, little is known about the role of boundary conditions in the self-organization of confined active fluids, especially fluid boundaries such as water–oil interfaces. Fluid boundaries are known to induce the coupling of the fluid dynamics on both sides of the boundary owing to hydrodynamic coupling27. For example, when active fluid is confined in a droplet immersed in liquid crystal, the liquid crystal develops oscillating rings surrounding the droplet31, which indicates that the active fluid alters the passive fluid configuration on the opposite side of the boundary. However, the principles underlying such coupling and the associated fluid mechanics have not yet been elucidated. Understanding such coupling dynamics is essential to unravel the dynamic role of fluid boundaries at interfaces between passive and active fluids and their impact on active fluid flows. Here, we investigated the hydrodynamic coupling between active and passive fluids in a water-in-oil active fluid droplet system with an approach that combines experiments and modeling. In our experiments, we confined an active fluid in a water-in-oil droplet that was compressed into a cylinder-like shape32 and characterized the flow coupling between active fluid and oil (passive fluid) near water–oil interface. We focused on how active fluids drive the oil through interface and on how the oil configuration can, in return, influence the self-organization of active fluid. We also determine the characteristic length scale of this active–passive fluid coupling. To gain deeper insight into our experimental results, we developed a continuum complex fluid simulation based on established active fluid models26 and explored methods of directing active fluid flows with novel millifluidic devices that can manipulate interfacial dynamics and droplet shapes in real time.Active fluids flow without external energy input owing to force generated by active entities that consume local fuel to generate kinetic energy37. Microtubule–kinesin active fluid is driven by extensile microtubule bundles that are assembled by depletion and extended by kinesin motor clusters , which causes flows. In this paper, we study the fluid dynamics of this active fluid when confined in a fluid cylindrical boundary.To investigate coupling between active and passive fluids across a fluid boundary, we selected microtubule–kinesin complex active fluid because it is tunable and reproducible and has established characterizations and models to serve as referencesers Fig. a38. The 39. However, the compressed droplets were motile40, so we curved the top plate into a dome-like shape to fix the droplet at the dome center and thus allow the long-term observation of intradroplet flows  as the average fraction of the azimuthal component in the flow velocity: COP ≡ mentclass2pt{minimTo investigate how the intradroplet flows were influenced by the shapes of the droplet and oil, we first immersed compressed active droplets with the same height (entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaR) or the thickness of the oil layer surrounding the droplet (Δ ≈ 0–11 mm) and measured the COP as a function of oil layer thickness ; oil layers thicker than this limit did not influence intradroplet flows. We hypothesized that the critical thickness of the oil layer originates from active fluid–oil coupling near the interface, which should not be sensitive to droplet shape40. To test this hypothesis, we repeated the measurements with droplet shapes with different r and h values  developed circulatory flows, the microtubule bundles near the water–oil interface tended to align at an angle ~ 15° from parallel with the interface . To distinguish the flows in both regimes, we doped the oil with 1-µm tracers and doped the droplet with 3-µm tracers  as well as in the droplet 27. Our data showed that when the droplet was in a circulating state  along with associated interfaces, but it also succeeds in describing the self-propelling and self-rotating characteristics of active droplets26. Moreover, Gao et al.’s model shows that active fluid encapsulated in a water-in-oil droplet can induce flows in the surrounding oil, which was observed in our experiments  active stress exerted by extensile microtubule-based bundles in active fluid that was proportional to the orientational order of bundles, 52, where 53; 54:u) that satisfied the Navier–Stokes equation:p is the fluid pressure, and The model considered two main forces: 1) interfacial tension force from the droplet surface,  interfacu, we initialized the flow field as quiescent fluids 55. Then we evolved the fluid flows for 3 h with the finite element method based on the computational fluid dynamics software COMSOL Multiphysics™57. We made the assumption that the geometry of the interface remained invariant over time and water–oil interfacial fluctuation was negligible, based on our experimental results , L is the correlation length), which suggests that the active fluid–oil interaction is short ranged  and had one movable wall  at the dome center . The milled chip was then cleaned with sequential 10-min sonication in detergent (Sigma-Aldrich Z805939), ethanol, and 100 mM potassium hydroxide solution and glued to a fluorophilically-treated glass slide (VWR GWBJ17) with epoxy (Bob Smith Industries BSI-201) to complete the device fabrication68.To confine the active fluid in a cylinder-like water-in-oil droplet, we designed a millifluidic device that compressed the droplet between a pair of plates separated by a height (ℎ) of 1 to 2 mm. Because the compressed droplet was self-propelling69 and pipetted the active fluid through the loading channel into the chamber of the fabricated millifluidic device that was filled with oil  depended on the ceiling–floor separation of the chamber and whose radius (r) depended on the pipetted fluid volume. After injecting the active droplet, we sealed the channel with epoxy. However, after the channel was sealed, air bubbles sometimes formed in the sample and affect the experimental outcomes. To keep the bubbles away from the droplet, we tilted the sample to direct the air bubbles to exit the chamber into the loading channel through the neck. The neck’s smaller opening prevented the bubbles from reentering the chamber  to cover the chamber. When the chamber was smaller than the ceiling (2R < 2Rb), the ceiling was trimmed to fit into the chamber; conversely, when the chamber was larger than the ceiling (2R > 2Rb), the ceiling was extended horizontally to match the chamber size , which could be imaged with a green fluorescent protein filter cube . To characterize the flow behaviors, we imaged the tracers for 3 h and tracked their trajectories To characterize flows in oil, we doped the oil with 0.002% Alexa 488-labeled 1-µm tracer particles (Polysciences 18860-1) and conducted the same observations and analyses for active fluid flows Fig. b, c. To ace Fig. c, d.69), excited with a 633-nm helium–neon laser, and observed in a 633–647 nm window. To reveal the microtubule arrangement in a circulating active fluid, we used our data to select the droplet shape  and oil layer thickness . The microtubules were labeled with Alexa 647  to remain flat (pink components in Supplementary Fig. To change the thickness of oil surrounding a droplet in real time, we designed a fluidic device with a movable wall, inspired by the mechanical iris that is used to adjust the aperture size of optical devicesTo control the shape of the droplet in real time, we designed a millifluidic device whose ceiling could be tuned manually. To fabricate the device, we separated the ceiling from the rest of the device and attached a handle to manually move the ceiling vertically. We also attached a hanger to hold the ceiling on top of the device chamber (Supplementary Fig. Supplementary Information 1.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.Supplementary Video 4.


Funding statement.In the original article, there was an error in the Funding statement. The funders “State Bureau of Investigation (AIE), and European Regional Development Fund (FEDER) (grant PGC 2018-096958-B-I00)” were mistakenly omitted. A correction has been made to the “This work was supported by the Spanish Ministry of Economy and Competitiveness (grant AGL 2013-45339R), the Spanish Ministry of Science, Innovation and Universities (MCIU), State Bureau of Investigation (AIE), and European Regional Development Fund (FEDER) (grant PGC 2018-096958-B-I00), and the Catalonian government (grant 2017SGR499). MG was recipient of a FPU grant from the Spanish Ministry of Science and Innovation.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.