The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected].


The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n = 61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n= 14), 91% of cases of CIN 2 (n= 11) and 67% of cases of CIN 3 (n= 15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n = 14) and CIN 2 (n = 10) and 11% false negatives for CIN 1 (n = 9) and 14% of false positives for normal cases (n = 21). The results of this preliminary study with reference to automation are discussed.


Objective: We undertook a microbiological study of purulent specimens from women with symptomatic breast abscesses.Methods: Fifty-one purulent samples were collected in 2 periods (December 1991–April 1992 and January 1994–June 1994) from nonpuerperal breast abscesses in 44 patients attending our hospital.Results: One of the most frequently isolated microorganisms was Proteus mirabilis , present as a pure culture in all but 1 specimen (isolated together with Peptostreptococcus spp.). Staphylococcus aureus was isolated in 10 specimens, 6 of which were post-tumorectomy abscesses. Polymicrobial anaerobic flora were isolated in 11 specimens (21.5%); Staphylococcus epidermidis in 4 (8%); and Streptococcus milleri, Alcaligenes sp., and mixed aerobic-anaerobic flora in 1 specimen each. The 7 remaining samples (13.7%) were negative bacteriological cultures.Conclusions: We draw attention to the frequent isolation of P. mirabilis in recurrent and torpid breast abscesses in 4 women in whom surgery was necessary in addition to antibiotic treatment.


Surveillance in the context of malaria elimination will needs to shift from measuring reductions in morbidity and mortality to detecting infections (with or without symptoms). The malaria elimination surveillance research and development agenda needs to develop tools and strategies for active and prompt detection of infection. The capacity to assess trends and respond without delay will need to be developed, so that surveillance itself becomes an intervention. Research is needed to develop sensitive field tests that can detect low levels of parasitaemia and/or evidence of recent infection. Examples of recent work on surveillance and response issues in several African countries will be discussed to illustrate approaches in active case detection and case investigations, cell phone reporting and response, and strategies to access mobile populations.


This case demonstrates very late neurological deterioration due to a pseudarthrosis in the fusion mass after scoliosis surgery. Though not the first case in the literature, it is the first case in which pre-operative magnetic resonance imaging revealed that the compression was due to a cyst arising from the pseudarthrosis.Twenty-two years after a successful correction and fusion for scoliosis, a 38-year-old Caucasian man presented with progressive numbness and significant weakness. As revealed by imaging, a cyst relating to an old pseudarthrosis was compressing the spinal cord. This was removed, and the cord decompressed, resulting in resolution of all symptoms.Lifetime care of patients with scoliosis is required for very late complications of surgery. Asymptomatic pseudarthroses have the potential to cause symptoms many years after surgery. This case report highlights a case of spinal cord compression and clinical neurological deterioration caused by a degenerative cyst arising from a pseudarthrosis in an area of previous scoliosis fusion 22 years after the index surgery. The case highlights the need for long-term follow-up of patients with scoliosis and for patients to have access to scoliosis services to address problems that may arise many years after the original surgery.The patient was a Caucasian man who was born in 1974 and who was first referred to the Spinal Deformity Service in 1977. He was initially treated with a brace for his mild scoliosis and followed up over many years until the age of 15, when his scoliosis had progressed, despite bracing, to a 44° curve between T8 and L3. Quite a rapid deterioration had occurred through the adolescent growth spurt, and consequently the patient underwent a posterior Harrington-Luque spinal fusion from T7 to L3. Bone graft was taken from the right iliac crest to create the fusion , and was a family man bringing up young children.In the summer of 2011, the patient, then 38, fell while playing paintball and injured his shoulder. The injury was musculoskeletal and settled reasonably quickly. However, in the two weeks that followed, he started to note increasing left leg weakness that was associated with some reduced sensation. The paraparesis progressively worsened to the point that he was unable to walk without sticks or a wheelchair. There had not been any disturbance in bowel or bladder function. The patient now reported altered sensation down the left side of his body from the L1 dermatome. In a neurological examination of his left leg, his motor power was significantly reduced: he had, at best, 3/5 power but most myotomes were of strength between 1/5 and 2/5 on the Medical Research Council grading scale. There was also blunting of sensation from the L1 dermatome distally.Spinal radiography at this time showed a good position of the Harrington rod and Luque wires as placed at the index surgery and a reasonable spinal alignment in both planes. Whole-spine magnetic resonance imaging (MRI) revealed a large cervicothoracic syrinx without Arnold-Chiari malformation. This was assumed to be old and not relevant to the current presenting complaint.The MRI also showed, within the area of instrumentation, a large cyst-like structure causing significant cord compression and this was presumed to be the cause of the recent neurological deterioration Figures and3. CThe patient underwent urgent surgery to decompress the spinal cord. Instrumentation using a modern pedicle screw system was placed to bridge the level of the proximal pseudarthrosis to provide stability. At the level of the compression on the cord, the fusion mass and pseudarthrosis were then burred away to reveal the spinal canal and cyst, the latter of which was removed and sent for a histological examination. The pseudarthrosis was repaired by using an iliac crest bone graft from the left posterior superior iliac spine (the side opposite that of the index procedure). After surgery, there was a wound breakdown with infection. This was managed through multiple debridements and vacuum-assisted closure therapy followed by a musculocutaneous flap to close the defect with a good result. The histological examination of the cyst revealed a degenerative cyst with no signs of malignancy or infection.At last follow-up at six months after surgery, the patient had full recovery of neurological function with no motor weakness or sensory loss and was returning to a more active life. Post-decompression MRI scans are unreadable because of the metal artifact of the Harrington rod and pedicle screw instrumentation over the area in question Figure. A CT scThis patient had, for the time, a well-performed operation with good correction of deformity and a stable construct which allowed him to return to a full and active life for 20 years without problems. It is worth noting that, although there were two separate pseudarthroses in the original fusion mass, both were asymptomatic until a reasonably minor trauma in the summer of 2011. Whether the degenerate cyst from the upper pseudarthrosis would have been present anyway or was stimulated to form after this trauma will never be known. The histology report revealed nothing more than degenerative and inflammatory changes.Neurological compromise secondary to pseudarthrosis within a previous posterior Harrington rod fusion at some time after the index surgery has been reported previously-4. HowevThe underlying cervicothoracic syrinx is undoubtedly related to why the spinal deformity occurred in the first place but is thought to be unrelated to the acute neurological compromise seen in this case as it was a lower-limb only problem with no symptoms in the upper limbs and the changes seen on the MRI appear to be longstanding.This case highlights the need for long-term scoliosis care for patients who have had scoliosis correction, fixation, and fusion in the past. Pseudarthrosis may not be apparent on plain X-ray, and the combination of CT scanning and MRI may be required to make the diagnosis given the difficulties in imaging older implants in an MRI scanner. The degenerative cyst that formed in this case was secondary to the micromovement of the pseudarthrosis, and the location in the spinal canal then caused neurological compromise. Although an operation on an asymptomatic pseudarthrosis may not be appropriate, patients need to be aware of the potential complications of leaving a pseudarthrosis alone given the late complication demonstrated in this case.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CT: Computed tomography; MRI: Magnetic resonance imaging.The author declares that he has no competing interests.


Exopolysaccharides (EPSs) produced by diverse group of microbial systems are rapidly emerging as new and industrially important biomaterials. Due to their unique and complex chemical structures and many interesting physicochemical and rheological properties with novel functionality, the microbial EPSs find wide range of commercial applications in various fields of the economy such as food, feed, packaging, chemical, textile, cosmetics and pharmaceutical industry, agriculture, and medicine. EPSs are mainly associated with high-value applications, and they have received considerable research attention over recent decades with their biocompatibility, biodegradability, and both environmental and human compatibility. However, only a few microbial EPSs have achieved to be used commercially due to their high production costs. The emerging need to overcome economic hurdles and the increasing significance of microbial EPSs in industrial and medical biotechnology call for the elucidation of the interrelations between metabolic pathways and EPS biosynthesis mechanism in order to control and hence enhance its microbial productivity. Moreover, a better understanding of biosynthesis mechanism is a significant issue for improvement of product quality and properties and also for the design of novel strains. Therefore, a systems-based approach constitutes an important step toward understanding the interplay between metabolism and EPS biosynthesis and further enhances its metabolic performance for industrial application. In this review, primarily the microbial EPSs, their biosynthesis mechanism, and important factors for their production will be discussed. After this brief introduction, recent literature on the application of omics technologies and systems biology tools for the improvement of production yields will be critically evaluated. Special focus will be given to EPSs with high market value such as xanthan, levan, pullulan, and dextran. Biopolymer  is used as a term to describe polymers produced by biological systems and polymers that are not synthesized chemically but are derived from biological starting materials such as amino acids, sugars, and natural fats . In nature, they have a significant role for protection of the cell, adhesion of bacteria tosolid surfaces, and participating in cell-to-cell interactions  and some non-carbohydrate substituents   that were able to produce two different polysaccharides have been reported , N-acetylgalactosamine , or glucuronic acid (GlcA) are the repeating units of heteropolysaccharides and occasionally non-carbohydrate substituent such as phosphate, acetyl, and glycerol. Homopolysaccharides and heteropolysaccharides are also dissimilar in synthetic enzymes and site of synthesis. Biosynthesis of homopolysaccharides requires specific substrates like sucrose, while the residues of heteropolysaccharide are produced intracellularly and precursors are located across the membrane by isoprenoid glycosyl carrier lipids for extracellular polymerization  ratio, oxygenation rate, and carbon sources can impact EPS production. EPS composition can be altered by conditional changes (differing monosaccharides or by monosaccharide molar ratio). For instance, due to the carbon source, Due to their unique and complex chemical structures that offers beneficial bioactive functions, biocompatibility, and biodegradability, microbial EPSs have find a wide range of application areas in chemical, food, pharmaceutical, cosmetics, packaging industries, agriculture, and medicine in which they can be used as adhesives, absorbents, lubricants, soil conditioners, cosmetic, drug delivery vehicles, textiles, high-strength materials, emulsifiers, viscosifiers, suspending, and chelating agents. In recent years, several novel bacterial EPSs have been isolated and identified; however, a few of them have achieved to have significant commercial value due to the high production costs  are also known as polysaccharide producers and one of the commercial EPS dextran producer Leuconostoc mesenteroides is a LAB; however, low production yields avoid LAB species to be exploited commercially. Besides, lactobacilli are GRAS  bacteria and their EPS could be utilized in foods   to glucose-6-phosphate (Glc-6-P). They are also involved in other cellular metabolisms. The second group is required to catalyze conversion of sugar nucleotides. Uridine-5′-diphosphate (UDP)-glucose pyrophosphorylase that catalyzes the conversion of Glc-1-P to UDP-Glc, which is one of the key molecules in EPS synthesis can be given as an example for this class of enzymes. Another enzyme group is glycosyltransferases (GTFs) that are located in the cell periplasmic membrane. The sugar nucleotides are transferred by GTFs to a repeating unit attached to glycosyl carrier lipid. The enzymatic functions, the structures, and identification of the genes that encode GTFs has been investigated intense and due to amino acid sequence similarities more than 94 GTF families were reported in the Carbohydrate-Active EnZymes (CAZy) database  transporter-dependent pathway, the synthase-dependent pathway, and the extracellular synthesis by use of a single sucrase protein. Inside the cell, the precursor molecules are transformed by enzymes and produce activated sugars/sugar acids in the first three mechanisms. Alternatively, in extracellular production pathway by direct addition of monosaccharides obtained by cleavage of di- or trisaccharides, the polymer strand is elongated , EPS biosynthesis and export have been reported to occur via the Wzx/Wzy-independent and Wzx/Wzy-dependent pathway  pathway, polymerization occurs at the cytoplasmic side of the inner membrane. The genes, which are required for high-level polymerization and surface assembly, are described as wza (encoding an outer-membrane protein), wzb (encoding an acid phosphatase), and wzc (encoding an inner-membrane tyrosine autokinase). In most Gram-negative bacteria -linked glucose monomers] or bacterial cellulose [β-(1-4)-linked glucose units] Rehm, . RegulatIn extracellular synthesis, polymerization reaction occurs as transfer of a monosaccharide from a disaccharide to a growing polysaccharide chain in the extracellular environment. This type of production of EPSs is uncomplicated; independent of the central carbon metabolism besides there is a limited variation in structure. The extracellular EPS synthesis can occur for homopolysaccharides  by extracellular GTF -rhamnose] regular repeating units from sugar nucleotide precursors, which are also involved in the biosynthesis of several cell wall components and can therefore be considered essential for growth  catalyze heteropolysaccharide biosynthesis which has numerous intracellular steps and only the last step that polymerization of repeating units occurs is extracellular. Depending on substrate type, uptake of sugars is achieved through a passive or an active transport system by the cell in the first step. Subsequently, the substrate is catabolized in the cytoplasm through glycolysis and sugar nucleotides are formed. The biosynthesis of activated precursors , which are derived from phosphorylated sugars is occurred. Finally, EPS is secreted to extracellular environment therefore their secretion from cytoplasm through cell membrane without compromising the critical barrier properties is a challenging process  and inulosucrases (EC 2.4.1.9) from transfructosidases class produce levan and inulin type fructans. Ftf genes are induced under stress conditions and sucrose hydrolysis and fructosyl transfer on fructan polymerized chain or syntheses of tri- or tetrasaccharides are catalyzed. In fructan, glucose is the non-terminal reducing residue (G-Fn)  and dissimilar to classical Leloir-type GTF they utilize sucrose as donor substrate instead of nucleotide-sugars. The transfer of monosaccharides, generating a glycosidic bond, from activated molecules to an acceptor molecule is catalyzed by these enzymes. Energy released by degredation of sugars is used to catalyze transfer of a glycosyl residue on forming polysaccharide. Due to the product of biosynthesis, the enzymes can be differentiated between transglucosydases (EC 2.4.1.y) and transfructosydases (EC 2.4.1.y or 2.y). Transglucosidases class includes dextransucrase, mutansucrase, and reuteransucrase (EC 2.4.1.5), which are high molecular weight extracellular enzymes and catalyze hydrolysis of sucrose to glucose and fructose and glucosyl transfer on carbohydrate or non-carbohydrate compounds. EPS structures can be varied based on different enzymes intervention and the synthesis of each polysaccharide is catalyzed by a specific GTF; therefore, two products encoded by two genes of Systems biology offers valuable application areas in molecular sciences, medicine, pharmacy, and engineering such as pathway-based biomarkers and diagnosis, systematic measurement and modeling of genetic interactions, systems biology of stem cells, identification of disease genes, drug design, strain development, bioprocess optimization  , polylactic acid (PLA), polysaccaharides, carboxylic acids, and butanediols. Systems biology approach and genome-scale metabolic models-guided metabolic engineering strategies have been successfully employed to enhance the productivity of useful biopolymers and their precursors , P (3HB-co-LA), by direct fermentation of metabolically engineered Pseudomonas putida was investigated by genome-scale metabolic model of this microorganism and survival under anaerobic stress was achieved by introducing the ackA gene from Pseudomonas aeruginosa and Escherichia coli  synthesizing capacity of Halomonas sp. TD01. In this study, several genes relevant to PHA and osmolytes biosynthesis were analyzed providing invaluable clues for understanding of the evolution and genes transfer, the strategic guidance of the genetic engineering of halophilic Halomonas sp. TD01 for co-production of PHA and ectoine.Cai et al.  reportedH. maura strain S-30 was performed to identify gene cluster in this strain. Three conserved genes, epsA, epsB, and epsC, also a wzx homolog, epsJ, which indicates that mauran is formed by a Wzy-dependent system, were found. It was also reported that eps gene cluster reaches maximum activity during stationary phase, in the presence of high salt concentrations (5% w/v), which was investigated by transcriptional expression assays using a derivate of H. maura S-30, which carries an epsA: lacZ transcriptional fusion  pathway is required, by performing in silico genome-scale metabolic analysis. Yim et al.  was identified  , which was reconstructed from genome data of, manually curated and further expanded in size. The impact of xanthan production was studied in vivo and in silico and compared with gumD mutant strain. This verified metabolic model is also the first model focusing on bacterial EPS synthesis and it can be used for detailed systems biology analyses and synthetic biology reengineering of Xcc. Moreover, draft genome of X. campestris B-1459, which was used in pioneering studies of xanthan biotechnology, and it can be used to analyze the genetic basis of xanthan biosynthesis has been reported recently  linkages between fructose rings. It is synthesized by the action of a secreted levansucrase (EC 2.4.1.10) that directly converts sucrose into the polymer  related to levan biosynthesis mechanism. The findings manifested mannitol as a significant metabolite for levan biosynthesis, which was further verified experimentally. In the previous study, 1.844 g/L levan yield from the stationary phase bioreactor cultures using a defined media containing sucrose as sole carbon source with almost fourfold increase of levan production was reported , and osmoprotectants. Genes related to EPS biosynthesis and intracellular PHA biosynthesis were detected. Hs_SacB gene encoding the extracellular levansucrase enzyme (EC 2.4.1.10), Pel polysaccharide gene cluster , Alginate lyase precursor (EC 4.2.2.3), and “Alginate biosynthesis protein Alg8” genes were predicted. The genome information and metabolic model will have a significant role on levan biosynthesis since they will be utilized to improve levan production by metabolic engineering strategies and medium optimization.Recently, Diken et al.  performeAureobasidium pullulans , UDP-glucose pyrophosphorylase , and glucosyltransferase . Nitrogen is the major component for the cultivation of A. pullulans N3.387 by ethyl methane sulfonaten (EMS) and ultraviolet (UV) mutagenesis to improve pullulan biosynthesis and developed a mutant that could produce more pullulan than wild type strain.Kang et al.  studied A. pullulans to understand the effect of different concentrations of (NH4)2SO4 which would be useful to optimize industrial pullulan production. The proteomic studies demonstrated the expression of antioxidant related enzymes and energy-generating enzymes and the depression of the enzymes concerning amino acid biosynthesis, glycogen biosynthesis, glycolysis, protein transport, and transcriptional regulation under nitrogen limitation, resulted in conversion of metabolic flux from the glycolysis pathway to the pullulan biosynthesis pathway.Sheng et al.  performeAureobasidium pullulans AY4 was determined and genome analysis revealed the presences of genes coding for commercially important enzymes such as pullulanases, dextranases, amylases, and cellulases , through the use of specific enzymes like glucansucrases  is part of a large EPS cluster, while dsrA (LEGAS_1012) is located as a single gene in the chromosome  LBAE C39-2, which was also found promising for in situ production of dextran in sourdoughs (Amari et al., Leuconostoc lactis EFEL005 has been announced and genomic analysis was performed to understand its probiotic properties as a starter for fermented foods (Moon et al., Various genome sequence and genome analysis studies were carried out with dextran producer strains. For instance, the genome of LAB In microbial EPS production, a better understanding of biosynthesis mechanism is a significant issue for optimization of production yields, improvement of product quality and properties, and also for the design of novel strains. As most of the novel bacterial EPS with unique properties have expensive production costs and economic hurdles need to be overcome, this valuable information about biosynthesis is also be important to lower these charges.More information on the genome of the EPS producer microorganisms will enable to develop additional strategies to successfully enhance EPSs production rate and also to engineer their properties by modifying composition and chain length. Since genome-scale reconstruction includes every reaction of target organism through integrating genome annotation and biochemical information, a systems-based metabolic modeling approach constitutes an important step toward understanding the interplay between metabolism and EPSs biosynthesis. Since microbial biopolymer biosynthesis is a result of a complex system of many metabolic processes, systems-based approaches are needed to control and optimize production in order to improve the formerly reported yields.Furthermore, the genome-scale metabolic model based on genome sequence will have the capacity to consider gene expression, metabolomics, and proteomics data to get accurate prediction at different environmental conditions.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacteria produce a wide range of exopolysaccharides which are synthesized via different biosynthesis pathways. The genes responsible for synthesis are often clustered within the genome of the respective production organism. A better understanding of the fundamental processes involved in exopolysaccharide biosynthesis and the regulation of these processes is critical toward genetic, metabolic and protein-engineering approaches to produce tailor-made polymers. These designer polymers will exhibit superior material properties targeting medical and industrial applications. Exploiting the natural design space for production of a variety of biopolymer will open up a range of new applications. Here, we summarize the key aspects of microbial exopolysaccharide biosynthesis and highlight the latest engineering approaches toward the production of tailor-made variants with the potential to be used as valuable renewable and high-performance products for medical and industrial applications. Polysaccharides produced by microbes can be generally classified by their biological functions into intracellular storage polysaccharides (glycogen), capsular polysaccharides which are closely linked to the cell surface  and extracellular bacterial polysaccharides  that are important for biofilm formation and pathogenicity. This article will focus on the latter, also termed EPS, which are secreted to the surrounding environment, and therefore can be efficiently harvested from cell-free culture supernatant in a continuous and cost-effective manufacturing process. At present four general mechanisms are known for the production of these carbohydrate polymers in bacteria: (i) the so called Wzx/Wzy-dependent pathway; (ii) the ATP-binding cassette (ABC) transporter-dependent pathway; (iii) the synthase-dependent pathway and (iv) the extracellular synthesis by use of a single sucrase protein. The precursor molecules, which are necessary for the stepwise elongation of the polymer strands, are realized by various enzymatic transformations inside the cell, and follow in principle the same concept of producing activated sugars/sugar acids in the first three cases of different biosynthesis pathways. For the extracellular production, the polymer strand is elongated by direct addition of monosaccharides obtained by cleavage of di- or trisaccharides.In the Wzx/Wzy dependent pathway individual repeating units, which are linked to an undecaprenol diphosphate anchor (C55) at the inner membrane, are assembled by several glycosyltransferases (GT’s) and translocated across the cytoplasmic membrane by a Wzx protein . In a next step their polymerization occurs at the periplasmic space by the Wzy protein before they will be exported to the cell surface . TranspoFigure 1). CPSs produced via this pathway all carry a conserved glycolipid at the reducing terminus composed of phosphatidylglycerol and a poly-2-keto-3-deoxyoctulosonic acid (Kdo) linker. This represents one of the main differences of the Wzx/Wzy and the ABC dependent pathways. Just recently novel insights into the early steps in CPS biosynthesis were provided by new discoveries of the structure of this conserved lipid terminus  biosynthesis . These pterminus .The third pathway is the synthase dependent pathway, which secretes complete polymer strands across the membranes and the cell wall, and is independent of a flippase for translocating repeat units. The polymerization as well as the translocation process is performed by a single synthase protein, which in some cases  is a subunit of an envelope-spanning multiprotein complex . SynthasTable 1).Most of the enzymatic steps for exopolysaccharide precursor biosynthesis appear inside the cell while polymerization/secretion is localized in the cell envelope. But there also exist some examples of extracellular synthesized polysaccharides, such as, e.g., dextran or levan. The biosynthesis of these occurs via GT’s, which are secreted and covalently linked to the cell surface . The genes involved in the different biosynthesis pathways encode various types of GT’s, polymerizing and branching enzymes, as well as enzymes responsible for addition of substituents or modifications of sugar moieties. Not all steps in the various pathways are currently understood, and sometimes the differences between the pathways become less defined. The genes encoding these enzymes can be found in most of the EPS producing microbes clustered within the genome or on large plasmids . Even though many gene clusters responsible for EPS biosynthesis have been known for several years, the function and mode of action of most of the genes and proteins is not completely clarified. An overview of the most relevant, commercial available EPS, including the biosynthesis pathway they are produced by is given in Table 1.In alignment with the various EPS biosynthesis pathways, the chemical structure and material properties of the final polymers are quite variable  to enhance the carbon flux toward the final polymer. In particular genes of precursor biosynthesis were overexpressed. This strategy was demonstrated to be successful for some EPS producers, but failed in some cases . AdditioAnother strategy of engineering EPS biosynthesis is aiming at tailor-made variants with desirable material properties for medical and industrial applications. Here the aim is to alter the molecular structure and therefore the behavior and material characteristics of the final polymer. For example these modifications can be based on deleting substituents or monomeric sugars from the side chain. On the other hand new or more substituents might be attached to change the ratio of decoration. Most efforts were done in engineering the degree of acetylation and pyruvylation of various polymers, in order to control their rheological behavior . Additioin vivo as well as in vitro approaches or even process parameters applied during the production process -linked glucose] and a side chain made of two mannose units and one glucuronic acid . Xanthan is produced by the two precursor’s glucose- and fructose 6-phosphate, the key intermediates of the central carbohydrate metabolism. At the moment five different genomes of X. campestris pv. campestris are available  was published which might further enhance the insights in conserved xanthan biosynthesis pathway  are not located within the gum-cluster. In detail, the assembly of the pentasaccharide repeating unit starts with the transfers of the first glucose unit toward the phosphorylated lipid linker (C55) anchored to the inner membrane via the priming GT GumD. In a next step, the cytosolic GT GumM attaches the second glucose unit by a β-(1-4)-bond to the first glucose. Catalyzed by GumH the first mannose unit is linked by an α-(1-3)-glyosidic bond, followed by the cytosolic glycosyltransferase activity of GumK which adds a β-(1-2)-linked glucuronic acid. Finally the repeating unit is completed by action of GumI, attaching the terminal mannose via a β-(1-4)-bond. In general most of the GTs involved in biosynthesis of EPS following the Wzx/Wzy–pathway appear to be monofunctional and the same applies specifically also for the xanthan biosynthesis  at which the repeat unit is still linked to the C55 anchor, which might play an important role in the targeted transport of the repeating unit .The general topology of Wzx proteins shows several transmembrane helices (TMHs), 10 in the case of GumJ . Data ofA putative adaption mechanism toward the length, as well as acceptance of repeating units with modified side chains was observed for some Wzy-proteins, characterizing them to be well suited for acceptance of tailored repeating units as obtained by genetic engineering .X. campestris genome  are placed on the highly conserved gene operon; the genes necessary for the other nucleotide sugar precursors are randomly distributed within the genome .Different heteropolysaccharides with closely related chemical structures, but strongly differing material properties belongs to the family of sphingans as produced by several strains, . The bacstrains,  with smastrains, . EPS incstrains, . The difstrains, . The gene genome . The asssed here . The assThe next steps are different in gellan, welan and diutan. Gellan represents the unbranched version of sphingans, which only shows substituent decorations of glycerol and acetyl at the second of the two glucose units of its backbone.L-rhamnose side chain attached to the first glucose residue of the growing repeating unit . The genes involved in incorporation of the side chains for welan and diutan have not been exactly functionally assigned at present. But the genes urf31.4, urf31, and urf34, which are labeled as “unknown reading frames” are assumed to be involved -linked rhamnose or mannose in the ratio of 2:1 as present at the first glucose of the repeat unit . Diutan ing unit  and two involved . These fvariants . Up to nf gellan .Whether the addition of the side chain sugars and non-sugar substituents occurs as final step of repeat unit assembly or at the nascent repeat unit remains speculative up to now, but as observed for xanthan, it can be assumed that already decorated repeat unit intermediates might reduce the activity of the GT’s involved in assembly of the repeat unit . The nexRhizobium, Agrobacterium, Alcaligenes, and Pseudomonas strains . Pyruvate is present in stoichiometric ratio, whilst succinate and acetate decoration depends on strain origin and culture conditions . The genes exoA, exoL, exoM, exoO, exoU, and exoW code for the GTs, subsequently elongating the octasaccharide repeating unit by addition of a glucose unit (Succinoglycan (SG) is an acidic EPS produced by several  strains . The modnditions . The pyrnditions  and nextnditions . Biosyntynthesis . ExoC, eectively . ExoY reose unit . ExoP  genes were identified to be involved in SG biosynthesis, R. melioti strains carrying mutated exsA (high similarity to ABC-transporter) variants showed an altered ratio of high molecular SG to low molecular SG, indicating involvement of exsA, without further knowledge of the detailed function. ExsB gene product was shown to have a negative influence on SG biosynthesis, resulting in lowered product titers  is localized on the same megaplasmid, but more than 200 kb away  also is known as the M antigen and is described to be an EPS . CA is mc amount . CA biosc amount . The genP-fucose . These tsynthase . The gensynthase . The nexsynthase . Due to synthase . The strsynthase .Figure 3). The Wzx protein was identified within the CA gene cluster by its typical transmembrane segments and the large periplasmic loop. WcaD is predicted to span the inner membrane with nine transmembrane segments, and to polymerize the repeat units of CA . Four genes are involved in curdlan biosynthesis . The curdlan synthase (CrdS), is the key enzyme of curdlan biosynthesis, showing a high similarity to cellulose synthases (Agrobacterium strains (Curdlan is a water insoluble β-(1-3)-glucan (glucose homopolymer) without any substituents, produced by, e.g., ynthases . The lacynthases . Many ge strains . Results strains . Curdlan strains . The bio strains . The Crd strains . First i strains . Additio strains .celS) genes from Acetobacter xylinum (acsABCD) was given in bacterial cytoplasmic membrane cellulose synthase (Bcs) proteins also belong to the GT2 family and are composed of three subunits -glucanase and the orf2 encoding for the “cellulose completing protein” are placed up-stream of the cellulose synthase operon  can be explained for the first time by the steric environment presented by the preceding glucose and the β-(1-4)-linkage, thus reversing the direction of terminal glucose rotation with every additional glucose monomer . These comonomers are arranged in blocks of continuous mannuronic acid residues (M-blocks), guluronic acid residues (G-blocks), or as alternating residues -linked β-tobacter .A. vinelandii alginates. However, alginates derived from pseudomonads do not contain G-blocks  and β-(1-3)-glycosidic bonds , causing β-hemolysis, as observed during cultivation of streptococci strains, recombinant HA production was of high priority even in the early stage of commercialization . Polymerization is realized outside the cell by the dextransucrase the key enzyme for dextran synthesis. Dextransucrases  belong to the enzyme class of transglucosidases that are part of the glucosyltransferases which are classified as glycoside hydrolase family 70  linked glucose . Levansucrases are widely distributed among Gram-positive bacteria and several plant pathogens carry more than one enzyme  and α-(2-1). Levan, which is produced by levansucrases, mainly consist of the former with occasional α-(2-1) branches. Inulin type polyfructan is obtained from inulinosucrase and shows the opposite α-(2-1) chain with α-(2-6) branches -dependent and cyclic di-GMP dependent regulatory mechanisms are available and will be summarized here.curdlan biosynthesis displayed up-regulation of GGDEF protein encoding genes under nitrogen limited conditions and lowered EPS production (57%) by knocking out c-di-GMP synthases  also includes nifR whose function is unknown. Knock-outs results in 30% reduction of curdlan production, whereas the knock-out experiments of σ-factor rpoN result in a 30% increased curdlan biosynthesis  resulted in a 70% decrease in curdlan production. Energy storage via polyphosphate influences curdlan biosynthesis as well as maintains the intracellular pH (crdR) is essential for curdlan production by operating as positive transcriptional regulator of the curdlan operon in ATCC31749. The potential binding region of crdR is located upstream from the crdA start codon , regulation  as well as its own operon  and seem to be mainly controlled by the Rcs (regulation of capsule synthesis) proteins, as reviewed by Factors inducing One of the main differences of CA regulation is the absence of CA production in wild-type strains grown at 37°C, whereas cultivation at lowered temperatures seems to induce CA biosynthesis . This tesuccinoglycan is occurring mainly at transcriptional and posttranscriptional level (exoS encoded membrane sensor together with the gene product of chvI (response regulator) constitute a two-component regulatory system which controls the expression of the exo genes. ExoR as a negative regulator directly acting on transcription and translation levels of most of the exo genes with exception of exoB , approaches implementing domain shuﬄing of GTs revealed to be of high potential to enhance the portfolio of EPS variants . The incin vivo as well as in vitro modification of EPS. The latter strategy was successfully applied to alginate and succinoglycan, both having in common the secretion of enzymes for EPS modification. Utilization of these secreted enzymes might allow a tight control of the material properties. Epimerases of A. vinelandii have been employed to modify alginate exhibiting a range of material properties (Alternatively protein engineering can be applied to EPS modifying enzymes for operties . Such vaoperties .Only recently an innovative bi-enzymatic process was reported for the production of short chain fructooligosaccharides and oligolevans from sucrose. This system was based on an immobilized levansucrase and an endo-inulase, resulting in a highly efficient synthesis system with a yield of more than 65% and a productivity of 96 g/L/h . The utiThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PLOS Computational Biology Education paper.This is a Research from educational psychology suggests that teaching and learning are subject-specific activities : learninThese tips will be useful to anyone teaching programming at any level and to any audience. A larger list aimed primarily at K–12 audiences can be found at [Guzdial  refers tThe most powerful evidence for this comes from Patitsas et al. . They exBeliefs such as this are known to have powerful effects on education outcomes –7. If inOne-on-one tutoring is perhaps the ideal form of teaching: all of a teacher's attention can be focused on one student, and they can completely customise their teaching for that person and tailor individual feedback and corrections based on a two-way dialogue with them. In realistic settings, however, one teacher must usually teach several, tens, or even hundreds of students at once. How can teachers possibly hope to clear up many learners' different misconceptions in these larger settings in a reasonable time?The best method developed so far for larger-scale classrooms is called peer instruction. Originally created by Eric Mazur at Harvard , it has 1. The instructor gives learners a brief introduction to the topic.2. The instructor then gives learners a multiple choice question that probes for misconceptions rather than simple factual recall. (A programming example is given in Code 1 that relates to integer comparison and loops.) The multiple choice question must be well designed. There is no point asking a trivial question that all students will get right or one with meaningless wrong answers that no student will pick. The ideal questions are those for which 40%–60% of students are likely to get the right answer the first time  3. Learners then vote on the answer to the question individually, thus formalising their initial prediction.4. Next, learners are given several minutes to discuss those answers with one another in small groups , and they then reconvene and vote again.5. Then, the instructor can act on the latest answers. If all the learners have the right answer, the instructor can move on. If some of the wrong answers remain popular after group discussion, the instructor can address those specific misconceptions directly or engage in class-wide discussion.Peer instruction is essentially a way to provide one-to-one mentorship in a scalable way. Group discussion significantly improves learners' understanding because it forces them to clarify their thinking, which can be enough to call out gaps in reasoning. Repolling the class then lets the instructor know if they can move on or if further explanation is necessary. While it significantly outperforms lecture-based instruction in most situations, it can be problematic if ability levels differ widely (as they often do in introductory programming classes because of varied prior experience). Pair programming (Tip 5) can be used to mitigate this.Rather than using slides, instructors should create programs in front of their learners . This is1. It enables instructors to be more responsive to "what if?" questions. While a slide deck is like a highway, live coding allows instructors to go off-road and follow their learners' interests or answer unanticipated questions.2. It facilitates unintended knowledge transfer: students learn more than the instructor consciously intends to teach by watching how instructors do things. The extra knowledge may be high level  or fairly low level .3. It slows the instructor down: if the instructor has to type in the program as they go along, they can only go twice as fast as their learners, rather than 10-fold faster as they could with slides—which risks leaving everyone behind.4. Learners get to see how instructors diagnose and correct mistakes. Novices are going to spend most of their time doing this, but it's left out of most textbooks.5. Watching instructors make mistakes shows learners that it's alright to make mistakes of their own . Most peLive coding does have some drawbacks, but with practice, these can be avoided or worked around:1. Instructors can go too slowly, either because they are not good typists or by spending too much time looking at notes to try to remember what they meant to type.2. Instructors can spend too much time typing in boilerplate code that is needed by the lesson but not directly relevant to it (such as library import statements). Not only does this slow things down, it can distract learners from the intended thrust of a lesson. As Willingham  says, "MNote that live coding does not always have to start with a blank screen: instructors may give students some starter code that relies solely on concepts they have already mastered and then extend it or modify it with live coding. Instructors who use live coding should ensure that learners have reference material available after lectures, such as a textbook, but should also recognize that students of all ages increasingly turn to question and answer sites such as Stack Overflow for information.When instructors are using live coding, they usually run the program several times during its development to show what it does. Surprising research from peer instruction in physics education shows that learners who observe a demonstration do not learn better than those who did not see the demonstration , and in The key to making demonstrations more effective is to make learners predict the outcome of the demonstration before performing it. Crucially, their prediction should be in some way recorded or public, e.g., by a show of hands, by holding up cue cards marked with A, B, C, or D, or by talking to their neighbour. We speculate that the sting of being publicly wrong leads learners to pay more attention and to reflect on what they are learning; regardless of whether this hypothesis is true, instructors should be careful not to punish or criticise students who predicted wrongly but rather to use those incorrect predictions as a spur to further exploration and explanation.Pair programming is a software development practice in which 2 programmers share 1 computer. One person  does the typing, while the other  offers comments and suggestions. The two switch roles several times per hour. Pair programming is a good practice in real-life programming  and alsoBoth parties involved in pair programming learn while doing it. The weaker gets individual instruction from the stronger, while the stronger learns by explaining and by being forced to reconsider things that they may not have thought about in a while. When pair programming is used, it is important to put everyone in pairs, not just the learners who may be struggling, so that no one feels singled out. It's also important to have people switch roles within each pair 3 or 4 times per hour so that the stronger personality in each pair does not dominate the session.Learning to program involves learning the syntax and semantics of a programming language but also involves learning how to construct programs. A good way to guide students through constructing programs is the use of worked examples: step-by-step guides showing how to solve an existing problem.Instructors usually provide many similar programming examples for learners to practice on. But since learners are novices, they may not see the similarity between examples: finding the highest rainfall from a list of numbers and finding the first surname alphabetically from a list of names may seem like quite different problems to learners, even though more advanced programmers would recognise them as isomorphic.Margulieux and Morrison et al. –21 have A principle that applies across all areas of education is that transference only comes with mastery . CoursesGuzdial et al. found that having learners manipulate images, audio, and video in their early programming assignments increased retention in 2 senses: learners remembered more of the material when retested after a delay and were more likely to stay in computing programs . This isA classic question in computing (and mathematics) education is whether problems are better with context  or without . Bouvier et al.  examinedOne caution about choosing context is that context can inadvertently exclude some people while drawing others in. For example, many educators use computer games as a motivating example for programming classes, but some learners may associate them with violence and racial or gender stereotypes or simply find them unenjoyable. Whatever examples are chosen, the goal must be to move learners as quickly as possible from "hard and boring" to "easy and exciting" .To help students accomplish a visible and satisfying result quickly, instructors can provide some prewritten software libraries or source code that starts students closer to the end goal. The idea that students must start from scratch and write all the code they need themselves is the relic of a bygone era of home microcomputers (and it was not true even then). Pick the task that you actually want to the students to engage in and provide everything else premade.This principle is tautological, but it is easily forgotten. Novices program differently than experts  and needNovices may need to spend time thinking about an algorithm on paper . They may need to construct examples in guided steps. They may struggle to debug. Debugging usually involves contrasting what is happening to what should be happening, but a novice's grasp on what should be happening is usually fragile.Novices do not become professionals simply by doing what professionals do at a slower pace. We do not teach reading by taking a classic novel and simply proceeding more slowly. We teach by using shorter books with simpler words and larger print. So in programming, we must take care to use small, self-contained tasks at a level suitable for novices, with tools that suit their needs and without scoffing.Our final tip for teaching programming is that you don't have to program to do it. Faced with the challenges of learning syntax, semantics, algorithms, and design, examples that seem small to instructors can still easily overwhelm novices. Breaking the problem down into smaller single-concept pieces can reduce the cognitive load to something manageable.For example, a growing number of educators are including Parsons Problems in their pedagogic repertoire , 27. Rathttp://sigcse.org/), ITiCSE , and ICER  present a growing number of rigorous, insightful studies with immediate practical application. Future work may overturn or qualify some of our 10 tips, but they form a solid basis for any educational effort to the best of our current knowledge.The 10 tips presented here are backed up by scientific research. Like any research involving human participants, studies of computing education must necessarily be hedged with qualifiers. However, we do know a great deal and are learning more each year. Venues like SIGCSE  or for gatekeeping . If you are teaching someone to program, the last thing you want to do is make them feel like they can't succeed or that any existing skill they have (no matter when or how acquired) is worthless. Make your learners feel that they can be a programmer, and they just might become one.Code 1. An example multiple choice question probing learners' understanding of loops and integer comparisonsfor  {if (i < 3 || i > = 8) {    System.out.println("Yes");        }    }How many times will the above code print out the word ‘Yes’?    a) 10    b) 5    c) 4    d) 3Code 2. An example of subgoal labellingConventional Materials    1. Click on "AccelerometerSensor1"    2. Drag out a when AccelerometerSensor1.AccelerationChanged block    3. Click on "cowbellSound"    4. Drag out call cowbellSound. Play and connect it after when AccelerometerSensor1.AccelerationChanged    Subgoal-Labelled Materials    Handle Events from My Blocks    1. Click on "AccelerometerSensor1"    2. Drag out a when AccelerometerSensor1.AccelerationChanged block    Set Output from My Blocks    3. Click on "cowbellSound"    4. Drag out call cowbellSound. Play and connect it after when AccelerometerSensor1.AccelerationChanged


American Muslim women are an understudied population; thus, significant knowledge gaps exist related to their most basic health behaviors and indicators. Considering this, we examined American Muslim women’s contraception utilization patterns.n = 224) met the inclusion criteria. Convenience sampling was employed. Multivariate logistic regression models estimated associations between demographics, marital status, ethnicity, nativity, health insurance, religious practice, and contraception use.Self-reported data collected in late 2015 were analyzed. Women who identified as Muslim, were at least 18 years old, sexually active, and current residents of the United States (Identifying as Muslim, in general, was significantly associated with greater odds of using contraception in general and condoms compared to American Muslim women who identify as Sunni. Identifying as Shia was associated with greater odds of using oral contraceptive pills relative to Sunni respondents. South Asian ethnicity was associated with higher odds of using oral contraceptive pills compared to those of Middle Eastern or North African ethnicity.Findings suggest American Muslim women’s contraception utilization patterns share certain similarities with both American women in general and disadvantaged racial and ethnic minority groups in the United States, implying that factors that influence American Muslim women’s use of contraceptives are possibly countervailing and likely multifaceted. More research is needed to accurately identify associates of contraceptive use in this population. This work serves as a starting point for researchers and practitioners seeking to better understand reproductive health decision in this understudied population. There is little scientific data available on the health-related knowledge, behaviors, and attitudes of American Muslim women. This knowledge gap is not surprising, considering American Muslim women are part of a religious minority population that experiences ongoing stigma; some American Muslim women are immigrants, and some are racial and ethnic minorities. To address this knowledge gap, we collected self-reported survey data from American Muslim women across the United States. We then statistically analyzed contraception utilization patterns of our respondents. Our study design and the variables included were informed by previous research on minority populations in the United States and social theory. We found that being a Shia Muslim or Muslim, in general (no sect declared) was significantly associated with higher odds of using contraception. Respondents identifying as South Asian had high odds of using oral contraceptive pills compared to Middle Eastern and North African respondents. Our findings suggest these American Muslim women’s contraception utilization patterns share similarities with American women in general and with disadvantaged minority groups. More research is needed to better understand barriers and facilitators of contraceptive use in this population.Muslim women in the United States are an understudied population; thus, significant knowledge gaps persist pertaining to their most basic health indicators and behaviors , 2. ThisNot only does contraceptive utilization vary across nations, it fluctuates widely across racial and ethnic groups in the United States –16. AmerOther theorists have examined factors which influence contraceptive use, and their theoretical frameworks suggest there are three behavioral factors that significantly influence the use of and type of contraceptive method selected: 1) Autonomous decision making authority of the woman, 2) Ability to negotiate with her health care provider  about contraceptive preferences, and 3) Influence of cultural subjective norms directly and indirectly related to contraception –23. WithNativity may also play a role in the decision to use contraceptives. The healthy migrant effect asserts that foreign-born individuals are healthier and more resilient than their American born peers; this is due to a selection bias in that only the healthiest individuals from a given population immigrate to the United States –32. ThisThe Muslim Women’s Health project was conducted for three months between September 2015 and December 2015. The goal of this study was to collect self-reported, exploratory data on a range of health behaviors and outcomes from American Muslim women. The primary outcomes for this particular sub-study related to contraceptive use; defined below. Respondents were recruited through online social networks, email requests, and postings made to online Muslim communities. To complete the survey or to learn more about this study. Information on the Muslim Women’s Health project, the funding institution , principal investigator, and ethical approval were available at this portal. Respondents were not compensated for their participation.N = 373. Our analysis of contraceptive utilization was limited to respondents who reported being sexually active (N = 224).Women who self-identified as Muslim, were at least eighteen years old, and were current residents of the United States were eligible to participate. Every respondent was asked to answer the same question in the same sequence -- question delivery was not randomized; respondents were able to skip any question, after the eligibility screening. Average time to complete the survey was about fifteen minutes due to the inclusion of a number of skip patterns allowing respondents to skip full sections that were not applicable. For example, if a respondent answered that she had never used any form of contraception; she was not asked questions about types of contraceptive used. Although online surveys have limitations, such as sampling bias, one major benefit is the ability to engage difficult-to-reach populations, including stigmatized populations, minority enclaves, and groups fearing persecution . Due to Our primary outcomes were use of contraception (any method), oral contraceptive pills, condoms, and the withdrawal method. Respondents were first asked if they had used contraception and then the type of contraceptive method was assessed by a 10-item multi-response question inquiring about the type of contraception used including: withdrawal method, condoms, oral contraceptive pills, intrauterine device, sponge, female condom, vaginal ring, diaphragm, contraceptive patch, and subdermal implant. Respondents who answered “yes” to contraceptive use were coded as 1 while respondents who answered “no” were coded as 0. Similarly, use of oral contraceptive pills, condoms, and the withdrawal method were assessed with binary variables. Respondents who answered “yes” were coded as a 1 and responses of “no” were coded as 0.To examine the relationship between individual characteristics and contraception use, we included independent measures of personal demographics, socioeconomic status, health insurance, and religion. Demographic characteristics were assessed four variables: age, marital status, ethnicity, and nativity. Age was operationalized with a continuous variable while marital status was assessed by a binary variable: married or not married. Ethnicity was operationalized with a series of binary indicators including Middle Eastern or North African, South Asian, and other (referent). Nativity was assessed with a single dummy variable, coded as 1 if the respondent was born in the United States and 0 if the respondent was born elsewhere. Socioeconomic status was assessed with two variables: annual household income grouped into six categories: under $24,999 (referent), $25,000–74,999, $75,000–99,999, and $100,000 and over, and education. Education was coded into four categories: high school education (referent), some college or vocational school, college graduate, and graduate or professional school. Health insurance was assessed with three categories: no insurance (referent), private insurance, and public insurance. Religion included two variables of religious sect and worship attendance. Religious sect was assessed with three categories: general Islam , Shia, and Sunni. Worship attendance included five categories: less than once a month (referent), once a month, multiple times a month, once a week, and multiple times a week.Frequencies described sexually active American Muslim women’s contraceptive use, demographics, socioeconomic status, religion, and health insurance status. Logistic regressions were used to estimate the relationship between demographics, socioeconomic status, religion, and the use and type of contraception. All analyses were conducted using Stata version 13.0.Ethical approval was obtained from University of Alabama at Birmingham’s Institutional Review Board . Informed consent was collected via an IRB approved online form.N = 224). The majority of respondents reported using contraceptives (79.5%). Approximately 66% used oral contraceptive pills (65.6%), 66.1% used condoms, and 32.1% used the withdrawal method. Average age was 32.2 years and ranged from 18 to 49 years. Two thirds of the respondents were married (66.5%). With respect to ethnicity, 44.2% of the respondents identifying as having South Asian ethnicity, 25.9% identified as having Middle Eastern or North African ethnicity, 29.9% identifying as having an ethnicity other than South Asian or Middle Eastern/North African. About 38% of the respondents were born in the United States (38.4%), 53.9% attended graduate or professional school, and 43.3% reported an annual household income of over $100,000. Most respondents carried private insurance (83.1%); only 5.6% were uninsured. About 38% (38.4%) identified as Sunni, compared to Shia (46.4%), and 15.2% identified as Muslim, in general (neither Shia nor Sunni). About 42% of the sample attended worship once a week or more frequently (42.34%).Table Table As to religious characteristics, respondents identifying as Shia were associated with 2.8 greater odds of contraceptive use, relative to respondents identifying as Sunni . Respondents identifying as Muslim, in general, were associated with 5.5 greater odds of using any form of contraception  and 3.7 greater odds of using a condom  compared to respondents identifying as Sunni. Worship attendance of multiple times a week was associated with lower odds of use of oral contraceptive pills, compared to respondents who report worship attendance of less than once a month . Respondents who reported worship attendance of once a month were associated with 6.0 higher odds of withdrawal than respondents who reported worship attendance of less than once a month .Certain non-statistically significant, but directionally relevant trends emerged, as well. Nativity did not have a statistically significant effect; however, American-born Muslim women had lower odds of using any contraception, oral contraceptive pills, and condoms. This reversed with utilization of the withdrawal method; with American-born women having higher odds to using the withdrawal method compared to their foreign-born peers. Education, although also not significant, did -- for the most part -- function as anticipated. Respondents with a graduate level of education utilized any contraception, oral contraceptive pills, and the withdrawal method at higher odds than respondents with a high school or lower level of educational attainment. Condom use was an outlier, with all higher levels of education having lower odds of condom use compared to the high-school referent category, possibly suggesting a bias against condom use in this particular population.Compared to the general public, racial and ethnic minorities have repeatedly been shown to have lower rates of contraceptive utilization and frequently use lower-efficacy methods than non-minority women , 13, 14.In contrast to the high rates of contraceptive utilization, we found a limited range of methods used; we found usage of oral contraceptive pills, condoms, and the withdrawal method. We found minimal utilization of the contraceptive patch, injectable contraception, and implants (statistics not reported). This gap between contraceptive methods employed in minority and non-minority women is growing and may allude to the impact of social and community circumstances that, with respect to American Muslim women, are unique to their culture and beliefs pertaining to reproduction , 38. ThuReligion and Islamic sect may influence the acceptability, perceptions, and attitudes of contraceptive use and may strongly influence the individual decision-making process. About 38% of our sample identified as Sunni Muslims. Shia respondents and respondents who identified as ‘general Islam’ -- a category we suspect may include those who are religiously more secular but culturally still identify as Muslim -- had higher odds of using contraception relative to Sunni Muslims. Considering Islam as a social modality, precise cultural criteria may define the reproductive practices within these societies and heavily influence the contraceptive choices acceptable to American Muslim women –41. ManyLimitations should be considered when applying these findings. Although our sample size is larger than other studies on Muslim health, it is still small compared to the population of Muslim women residing in the United States. Selection bias existed; respondents were more educated and wealthier than Americans in general, and we suspect even in comparison to the wider American Muslim population. Yet, it is noteworthy that these high income, highly educated American Muslim women had contraceptive utilization rates that outpaced contraceptive use found in the highest quintile of Muslim women in nations from which respondents migrated, such as Pakistan . AlthougGiven the complexities of delivering reproductive health care and increasing access to contraceptives within secular societies, it is likely that additional confounding arises when considering the cultural preferences of Muslim Americans . Prior s


Scientific Reports6: Article number: 3852910.1038/srep38529; published online: 12062016; updated: 05172017In Figure 1, the latitudes ‘80.5 N’ and ‘81.0 N’ were incorrectly given as ‘81.0 N’ and ‘81.5 N’ respectively. In addition, the scale between 0 km and 40 km was incorrectly given as between 0 km and 80 km. The correct Figure 1 appears below as


Domains of functional performance  and cognition  have been shown to independently predict both comfortable and gait speed. Walking speed reserve (WSR) reflects an individual’s ability to increase their walking speed when needed to adjust to environmental demands. Increasing speed requires a higher demand of neuromuscular control and cognitive resources. Few studies have investigated WSR; however, poorer cognitive status has been shown to be associated with a smaller WSR. Understanding mechanisms in WSR could help with intervention development. The purpose of this study was to investigate the interplay of functional performance and cognition on WSR. Sixty-seven community-dwelling older adults  completed assessments including: global cognition , leg strength (30 Second Chair Stand), balance , executive function , simple reaction time, processing speed (Digit Symbol Substitution Test), and gait speed (comfortable and fast) using the GAITRite® system. WSR (fast-comfortable gait speed) was the dependent variable. Independent variables in the regression model were selected using Pearson correlation values <0.05  and processing speed ). Linear regression revealed that the strength and processing speed explained 16.3% of the variance (adjusted r2) of WSR and were both unique predictors of WSR indicating individuals with greater strength  and faster processing speed  have a greater capacity to alter their gait speed. Further, the interdependence between cognition and physical performance emphasizes the importance of interprofessional care for older adults.


Here we address four issues associated with BBB models: cell source, barrier function, cryopreservation, and matrix stiffness. We reproduce a directed differentiation of brain microvascular endothelial cells (dhBMECs) from two fluorescently labeled human induced pluripotent stem cell lines (hiPSCs) and demonstrate physiological permeability of Lucifer yellow over six days. Microvessels formed from cryopreserved dhBMECs show expression of BBB markers and maintain physiological barrier function comparable to non-cryopreserved cells. Microvessels displaying physiological barrier function are formed in collagen I hydrogels with stiffness matching that of human brain. The dilation response of microvessels was linear with increasing transmural pressure and was dependent on matrix stiffness. Together these results advance capabilities for tissue-engineered BBB models.Three-dimensional (3D) tissue-engineered models of the blood-brain barrier (BBB) recapitulate  Brain microvascular endothelial cells (BMECs) form tight junctions, which restrict paracellular transport, and an array of efflux pumps and transporters, which regulate transcellular transport into the brain. The functional BBB prevents entry of the majority of pharmaceutical agents into the brain, particularly those of high molecular weight, hindering treatment strategies for CNS disease2, while a dysfunctional BBB is implicated in neurological disease4. BMECs generated via differentiation of human induced pluripotent stem cells (hiPSCs) are a promising cell source for in vitro studies of the BBB8. However, most models of the BBB are constructed in two-dimensions (2D) and are therefore unable to recapitulate shear stress, cell-ECM interactions, and cylindrical geometry characteristic of the in vivo brain microvasculature9. Three-dimensional (3D) models of the BBB that incorporate these complexities and achieve physiological barrier function have recently been developed12.Microvessels of the blood-brain barrier (BBB) separate the bloodstream from the central nervous system (CNS)in vivo. However, multiple challenges limit the application of such models: (1) The spontaneous differentiation of iPSCs remains a time-consuming step in microvessel fabrication. The differentiation generates a sub-population of BMECs which is purified by subculture on collagen IV and fibronectin-coated surfaces7. This purification step reduces cell adhesion and has restricted formation of microvessels to stiff collagen I hydrogels13. However, the use of cross-linkers to increase stiffness can limit the ability to co-culture cells in the surrounding matrix. As an alternative, the directed differentiation of dhBMECs5 does not involve a purification step and hence could increase the efficiency of microvessel fabrication and expand the range of matrix materials. Furthermore, extending the repertoire of cell lines to include fluorescently labeled hiPSCs is critical to enabling live-cell imaging in 3D tissue-engineered models. (2) Mimicking physiological barrier function in tissue engineered models is critical for studying clinically-relevant processes such as BBB-opening and drug delivery15. While recent advances have led to in vitro models with physiological barrier function; the ability to maintain stable barrier function within tissue-engineered models has not widely been reported10. This limits the utility of models for studying processes which occur over days to weeks. (3) dhBMECs do not maintain phenotype following passaging, therefore, freshly differentiated dhBMECs are required for most experiments. Recent studies have reported that cryopreservation of dhBMECs obtained by spontaneous differentiation16 show similar barrier properties as freshly differentiated cells. Cryopreservation has not been validated for 3D BBB models, but would increase fabrication efficiency. (4) As the human brain is highly cellular (70–85% by volume), selection of an appropriate extracellular matrix (ECM) for 3D models is challenging17. As a proxy for the cellular components, 3D models of the BBB commonly utilize ECM proteins not present in the brain, including collagen I22 and fibrin23. In previous work we demonstrated physiological barrier function in a tissue-engineered microvessel model within a stiff collagen matrix13. However, the role of matrix stiffness on the structure and function of 3D blood-brain barrier microvessels has not been further explored.Tissue-engineered models are indispensable for studies of the BBB as they integrate spatial and temporal information on cell behavior and barrier function, similar to two-photon microscopy approaches 5 using fluorescently-tagged hiPSCs which enable live-cell imaging, (2) barrier function – we demonstrate stable and physiological permeability of solutes over six days, (3) cryopreservation – fresh and cryopreserved dhBMECs display similar barrier function in 2D and 3D models, and (4) the role of matrix stiffness in BBB structure and function – the solute permeability of microvessels remains low across matrix stiffness ranging from 0.3–3.3 kPa, while the structural stability and dilation response were stiffness-dependent. Together, these advances in cell culture and live-cell imaging establish tissue-engineered microvessels as a versatile platform for studies of BBB function/dysfunction and highlight the importance of matrix stiffness in BBB microvessel behavior.In this paper we generate tissue-engineered 3D models of the human BBB which address these key challenges: (1) cell source – we reproduce a recently reported directed differentiation of dhBMECs5 for the directed differentiation of two fluorescently-labeled human induced pluripotent stem cell lines (hiPSCs), BC1-green fluorescent protein (BC1-GFP) and C12-red fluorescent protein (C12-RFP), into brain microvascular endothelial cells (dhBMECs). The directed differentiation uses a multistep protocol over the course of eight days , matching the concentration previously used for directed differentiation of BMECs   . BC1-GFP dhBMECs maintained TEER values above 2,000 Ω cm2 for seven days, and C12-RFP dhBMECs maintained TEER values above 1,500 Ω cm2 for two days . Retention of physiological barrier function is critical for studies requiring long incubation periods, such as studies of neurodegenerative protein aggregate accumulation.On day eight of the differentiation, dhBMECs were singularized and seeded onto Matrigel-coated Transwells to monitor TEER Fig. . TEER peely Fig. . The difays Fig. . Both liays Fig. . TEER is7. The relative contributions of endothelial markers and tight junction proteins on barrier function are not well understood; however, claudin-5 is thought to be critical to achieve physiologically high TEER values31.To determine whether cells properly express characteristic BBB endothelial markers, we stained confluent monolayers for tight junction markers, nutrient transporters, and efflux transporters Fig. . Directe−1 collagen gels13. This approach relies on the isolation of brain endothelial cells from a heterogeneous cell population through selective adhesion, achieved by sub-culturing terminally differentiated cells onto collagen IV and fibronectin-coated dishes prior to seeding into microvessels. To assess the barrier function of dhBMECs from the directed differentiation, we formed functional microvessels using a templating method reported previously  of 1–4 dyne cm−2 37.First, we assessed microvessels formed in 7 mg mLtep Fig. . Monolay3. To further evaluate barrier function, microvessels were simultaneously perfused with two different size fluorescent probes: Lucifer yellow (LY) and 10 kDa Dextran 38. Similar values were also obtained for BC1-derived dhBMECs and Madin-Darby Canine Kidney (MDCK) cells in 2D transwells, suggesting that 3D culture is not required to achieve physiological barrier function39.BBB microvessels expressed claudin-5 localized at cell-cell junctions Fig. . Claudinran Fig. . Permeabran Fig. . The perely Fig. . These v−7 cm s−1 due to photobleaching of hydrogel autofluorescence resulting from the cross-linker.11 Studies in zebrafish have shown that 10 kDa dextran does not enter the brain40; however, there remain discrepancies across in vivo studies concerning quantification of BBB permeability9.The permeability of 10 kDa dextran in both BC1-GFP and C12-RFP dhBMEC microvessels was below our detection limit Fig. , similar2 for seven days. To confirm that 3D microvessels retained barrier function over time, we repeated permeability measurements of BC1-GFP microvessels on day six. Lucifer yellow permeability in BC1-GFP microvessels was 5.02 ± 0.94 × 10−7 cm s−1 on day six, not statistically different from day two . In addition, the permeability of 10 kDa dextran remained below the detection limit, indicating preservation of barrier function. These results suggest that barrier function can be maintained in 3D models without need for co-culture with supporting cell types or additional factors. Recently, we found that the incorporation of pericytes into BBB microvessels does not reduce solute permeability41.In 2D, TEER values for BC1-GFP dhBMECs remained above 2,000 Ω cm16. A working stock of cryopreserved dhBMECs enables scale-up, higher throughput, and can reduce batch-to-batch variability between experiments. BC1-GFP dhBMECs, which maintained higher TEER values than C12-RFP dhBMECs, were cryopreserved in liquid nitrogen  for 11–120 days. The phenotype and barrier function of cryopreserved BC1-GFP dhBMECs was assessed and compared to non-cryopreserved controls using 2D and 3D functional assaysIn tissue engineering, cell cryopreservation can eliminate the need to perform unique differentiations before each experiment. Cryopreserved dhBMECs have been shown to maintain BBB phenotype when thawed in media containing Rho-associated protein kinase inhibitor Y27632 (ROCK inhibitor)2 for more than ten days . Cryopreserved dhBMECs also retained expression and localization of critical tight junction, efflux, and transporter proteins comparable to non-cryopreserved controls . The morphology of thawed dhBMECs was indistinguishable from newly differentiated cells upon seeding onto glass surfaces. Cryopreserved dhBMECs achieved peak TEER values three days after seeding into Transwells , while fresh cells typically peaked two days after seeding. Confluent monolayers of cryopreserved dhBMECs exhibited TEER values above 1,000 Ω cmays Fig. , and a p. 2 Fig. . This isols Fig. .Figure 5−1 collagen I gels cross-linked with genipin at the same density as microvessels formed with non-cryopreserved cells (20 × 106 cells mL−1). Microvessels with freshly differentiated dhBMECs reached confluence one to two days after seeding, whereas the time to confluence was two to three days with cryopreserved cells, possibly due to reduced cell proliferation. However, the resulting microvessel morphology was indistinguishable to those formed with non-cryopreserved dhBMECs. Barrier function was assessed from permeability measurements following microvessel formation. The permeability of Lucifer yellow in cryopreserved dhBMEC microvessels was 3.22 ± 0.77 × 10−7 cm s−1 . The permeability of 10 kDa Dextran was below the detection limit . Thus, to accurately model the brain ECM, either large volumes of cells must be incorporated or a relatively inert matrix material must be selected to provide sufficient structural support9. As a result, 3D models of the BBB are commonly generated using ECM proteins not found in the brain23. In previous work we used genipin cross-linked 7 mg mL−1 collagen I gels to form microvessels13.The ECM of the brain is comprised of hyaluronic acid, lecticans, proteoglycan link proteins, and tenascins49. The origins of this broad range are thought to be due to differences in brain region47, testing methods50, and preparation techniques51. Therefore, to enable direct comparison of our models and in vivo conditions, we obtained elastic moduli for mouse brain and hydrogels from stress - strain curves in compression53. We tested four matrix conditions: (1) 7 mg mL−1 collagen I cross-linked with genipin, (2) 7 mg mL−1 collagen I, (3) 5 mg mL−1 collagen I, and (4) 3 mg mL−1 collagen I.Previous measurements of brain tissue have reported elastic moduli between 0.5–4.5 kPa−1 cross-linked gels, 0.8 ± 0.2 kPa for 7 mg mL−1 gels, and 0.3 ± 0.2 kPa for 5 mg mL−1 gels. 3 mg mL−1 collagen I hydrogels were not sufficiently stiff for unconfined bulk mechanical testing. Genipin cross-linking increased the stiffness of 7 mg mL−1 gels by about 4-fold (p < 0.001), similar to previous reports54. For comparison, we measured the stiffness of resected mouse brain using the same method. The Young’s modulus for mouse brain was 2.1 ± 0.4 kPa, between values of 7 mg mL−1 and 7 mg mL−1 genipin cross-linked collagen I. Based on the elastic moduli, 7 mg mL−1 and 7 mg mL−1 genipin cross-linked collagen I are reasonable choices for emulating the stiffness of the brain.The Young’s modulus of the bulk gels increased with increasing collagen density Fig. . The mod49. Decreases in stiffness are observed during Alzheimer’s disease, but still are between values for 7 mg mL−1 cross-linked and non-crosslinked collagen I (2.40–2.51 kPa)48. The effect of changes in brain stiffness on blood-brain barrier structure and function is not known; however, changes in basement membrane are thought to alter brain function56.Changes in brain stiffness, as measured using magnetic resonance elastography (MRE), are possible biomarkers for disease and aging−1 collagen I gels cross-linked with genipin to form microvessels13. However, the use of genipin poses multiple challenges for tissue-engineering, including the difficulty in culturing cells within highly cross-linked matrices and the cytotoxicity of unreacted cross-linker57. Reliable formation of BBB microvessels without the use of chemical cross-linkers would expand model complexity.To assess the adhesion and barrier function of dhBMECs over a range of ECM stiffness, microvessels were formed from non-cryopreserved BC1-GFP dhBMECs. In previous work, we used 7 mg mL−1 collagen I but did not remain perfusable due to collapse of the endothelium   Fig. . The perely Fig. . As desc−1) and softest (5 mg mL−1) hydrogels in which stable microvessels were formed, the transmural pressure was increased by increasing the height between the fluid inlet and outlet reservoirs. We observed the response to 2-fold and 3-fold increases in transmural pressure above the baseline head of 5 cm H2O (approximately 0.05 Pa) . For microvessels in cross-linked 7 mg mL−1 gels, a 3-fold increase in transmural pressure resulted in a ~6% increase in diameter .To assess the dilation response of the stiffest (cross-linked 7 mg mLPa) Fig. . Dilatioter Fig. . For 5 mion Fig. . The res59. Relaxation and constriction of smooth muscle cells (SMCs) mediates these changes in the brain. Physiological arteriolar dilation ranges from about 10–30%; however, the diameter of these arterioles is typically less than 20 μm61. Autoregulation, the process by which tissue perfusion is maintained constant, is also controlled by changes in arterial diameter63. Autoregulation is maintained through multiple mechanisms including myogenic responses where vasoconstriction (mediated by SMCs) occurs in response to increases in transmural pressure (and vice versa)64. In arterioles of skeletal muscle in rats, a 3-fold decrease in perfusion pressure results in ~20% dilation (increase in diameter from ~85 μm to ~100 μm). However, when the SMC response is abolished (using treatment with Ca2+ free solution), a 3-fold decrease in perfusion pressure results in ~25% constriction (decrease in diameter from ~135 μm to ~100 μm)65. Additionally, in the absence of SMCs, changes in diameter were observed to be roughly linear in the same fold-change regime as studied here65. These studies suggest that our 5 mg mL−1 microvessels display dilation behaviors similar to in vivo arterioles devoid of SMCs. Incorporation of SMCs into tissue-engineered microvessels could recapitulate myogenic autoregulation of flow in response to transmural pressure changes.Neurovascular coupling, the process by which cerebral blood flow is matched to neuronal metabolic demand, is controlled by dilation of arterioles directly upstream from capillariesWe address four key challenges associated with fabrication of tissue-engineered BBB models: cell source, barrier function, cryopreservation, and matrix stiffness. Functional microvessels formed from dhBMECs obtained from the directed differentiation of two iPSC lines showed expression of BBB markers and maintain physiological barrier function. Similarly, functional microvessels with physiological barrier function were formed from cryopreserved dhBMECs, eliminating the need for performing differentiation prior to model fabrication. Microvessels with physiological barrier function were formed in gels with stiffness ranging from 0.3–3.3 kPa, spanning the stiffness of mouse brain (~2 kPa). The dilation response of microvessels was linear with transmural pressure and was dependent on matrix stiffness, enabling simulation of the cardiac cycle.66 line was derived from a 46 year old male and the C12-RFP67 line derived from a newborn male. Since the research does not involve humans or animals, and the iPS lines are de-identified, no IRB approval was required. hiPSCs were maintained between passages P50-P70 and were passed using Versene (ThermoFisher). Prior to differentiation, hiPSCs were singularized using warm Accutase (Invitrogen) and plated onto Matrigel-coated six-well culture plates in mTeSR1 supplemented with 10 μM Rho-associated protein kinase inhibitor Y27632  at a density between 10,000 and 50,000 cells cm−2  were maintained on Matrigel-coated tissue-culture treated six-well culture plates (Corning) in mTeSR1 medium (Stem Cell Technologies). Two iPSC lines were used in this study: the BC1-GFP5. Briefly, hiPSC colonies were expanded for three days in mTeSR1, then were treated with CHIR99021 (Selleckchem) at concentrations between 1–6 μM , 10 μM all-trans retinoic acid (Sigma) and 1 × B27. The medium was not changed for 48 hours.Directed differentiation of hiPSCs was adapted from a previously reported protocolhiPSC cultures were imaged daily using phase contrast microscopy and fluorescence microscopy using FITC or Texas Red filters. 10.2 mm × 7.65 mm images were acquired on an inverted microscope (Nikon Eclipse TiE) using a 4 × objective (Nikon) with epifluorescence illumination provided by an X-Cite 120LEDBoost (Excelitas Technologies).2 area, 0.4 µm pore size) were seeded at 1 × 106 cells cm−2, while eight-chambered borosilicate cover glass wells (Lab Tek) were seeded at 0.8 × 106 cells cm−2. All surfaces were coated overnight with 100 μg mL−1 growth factor-reduced Matrigel (Corning) in DMEM/F12. Cells were maintained in two-dimensional culture assays using hECSR1 for 24 hours then switched to hECSR2: hESFM supplemented with 1 × B27. Transwell cultures were maintained for 14 days, without further media changes. TEER was recorded daily as previously described6.On day eight of the differentiation, cells were singularized using warm Accutase for one hour on a shaker at 100 rpm. Corning® Transwell® polyester membrane cell culture inserts , then fixed using ice-cold methanol for 15 minutes, then blocked for 30 minutes in PBS with 10% normal goat serum  and 0.3% Triton X-100 (Millipore Sigma). Primary antibodies were diluted in blocking buffer and incubated on cells overnight at 4 °C  which served as a template for the microvessel. Collagen solutions were neutralized on ice and then gelled for 30 minutes at 37 °C. Prior to the removal of the template rod, the addition of 2% agarose to both sides of the collagen gel prevented delamination. To increase stiffness, 7 mg mL−1 gels were crosslinked using 20 mM genipin (Wako Biosciences) for two hours54; devices were then perfused with PBS for at least eight hours to remove excess genipin. Before seeding with dhBMECs, channels were coated overnight with 100 μg mL−1 growth factor-reduced Matrigel in DMEM/F12. Derived hBMECs were seeded at 20 × 106 cells mL−1 into collagen channels using hESCR1 media supplemented with 10 μM ROCK inhibitor Y27632 and allowed to adhere under static conditions for 30 minutes. ROCK inhibition was required to maintain cell adhesion, as previously reported13. After 24 hours, ROCK inhibitor was removed and microvessels were continually perfused with hECSR2. After cell seeding, microvessels were maintained under constant perfusion using a gravity driven flow system (inlet pressure of 5 cm H2O); the average endothelium shear stress was approximately 1 dyn cm−2 as calculated from Poiseuille’s Law.Microvessels were formed within a rectangular channel in a polydimethylsiloxane  housing with dimensions 1 cm (length) × 1.75 mm (width) × 1 mm (height) formed within an aluminum mold. The PDMS housing was attached to a glass slide following plasma treatment and treated with trimethoxysilane (Sigma) prior to gelling to reduce bubble formation between the PDMS and collagen gel. Neutralized rat tail type I collagen gel (Corning) ranging from 3 to 7 mg mLPermeability was measured using two different molecular weight solutes: 200 μM Lucifer yellow  and 2 μM Alexa Flour-647-conjugated 10 kDa dextran (Thermo Fisher). Phase contrast and fluorescence images were acquired every two minutes (NIS Elements) before  and after solute perfusion . At every time point, the protocol obtained six images: (1) a phase contrast image of the top of the microvessel (located and maintained by autofocus), (2–5) phase contrast and fluorescence images of the microvessel midplane, and (6) a phase contrast image of the bottom of the microvessel. Filter cubes (Chroma 39008 and Chroma 41008) were used to capture Lucifer yellow (20 ms exposure) and Alexa Fluor-647-conjugated dextran (200 ms exposure), respectively. Images were collected as ten adjacent frames using a 10× objective, resulting in a total image area of 8.18 mm × 0.67 mm.0, where d is the diameter of the vessel, ΔI is the initial increase in total fluorescence intensity, and (dI/dt)0 is the rate of increase in total fluorescent intensity69. The rate of intensity increase (dI/dt) was measured over sixty minutes for Lucifer yellow and thirty minutes for 10 kDa dextran. Images were segmented into 10 adjacent regions-of-interest (ROIs), where the value of the ROI with minimum permeability is reported to exclude artifacts from interstitial dye entering the ECM from the inlet and outlet ports. The permeability detection limit was 1 × 10−7 cm s−1, as previously reported11.ImageJ (NIH) was used to measure fluorescence intensity profiles over 36 frames for each of the ten adjacent frames. Permeability was calculated from (d/4)(1/ΔI)(dI/dt)The structure of BBB microvessels was monitored daily using phase contrast imaging. Survival of microvessels represents two criteria: (1) maintenance of perfusion, and (2) confluent endothelium attached to the collagen wall. Additionally, the ability of BBB microvessels to respond to fluid pressure was monitored using microscopy while the inlet and outlet height difference was changed. The inlet height was increased in increments of 5 cm from 5 cm to 15 cm and equilibrated for 5 minutes at each height. Phase contrast images were acquired at each pressure to record average microvessel diameter.16. dhBMECs were singularized in Accutase and then resuspended in 60% hESCR1, 30% fetal bovine serum (Sigma) and 10% dimethyl sulfoxide (Sigma). Cryovials were frozen in an isopropanol-filled freezing container  at -80 °C for 24 hours, then moved into liquid nitrogen for long-term storage. Cells were rapidly thawed in a water bath (~37 °C), centrifuged using fresh media, and then seeded onto transwells or glass, or seeded into microvessels as previously described. 10 μM ROCK inhibitor Y27632 (RI) was supplemented for the first 24 hours after thawing.Human iPSC-derived BMECs were cryopreserved and stored in liquid nitrogen on day eight as previously described−1 using a tensile/compression tester (MTS Criterion). From the obtained stress-strain curve, the Young’s Modulus (Pa) was calculated as the best-fitted slope of the initial linear region (~5–12% strain). Compression testing could not be accurately conducted on 3 mg mL−1 collagen I hydrogels as they were physically unstable when unconstrained and collapsed by ~40% (height). The hydrogels of other conditions were stable and their heights varied less than 10%.Hydrogels: Collagen hydrogels were fabricated by casting collagen I solutions between two glass plates separated by 2 millimeters. Collagen I solutions were gelled for 30 minutes at 37 °C. Some hydrogels were crosslinked with genipin and/or equilibrated with PBS as previously summarized. Hydrogel samples 10 mm in diameter and 2 mm in thickness were punched out and compressed at a rate of 0.25 mm s−1 using a tensile/compression tester (MTS Criterion). The Young’s modulus was calculated from the obtained stress-strain curve (~5–10% strain).Mouse brain: 9–12 weeks old male and female BALB/c mice were euthanized, and had their brains harvested within one hour of compression tests. A coronal section of the whole brain was taken by cutting at the anterior and posterior coronal plane proximal to the pituitary gland (~3 mm thickness). Once the sample was sectioned, it was immediately staged on the compression tester for measurement. The compression plate was lowered to achieve initial contact with the flat tissue surface with minimum compressive force applied. The initial thickness of the tissue was then measured by the distance between the top and bottom (stage) plates for strain calculation. The sample was allowed to stabilize for one minute before compression testing. The tissue sample was then compressed at a rate of 2 mm minStatistical testing was performed using Prism ver. 8 (GraphPad). TEER, permeability and Young’s moduli measurements are reported as mean ± standard error of the mean (SEM). A student’s unpaired t-test  or an analysis of variance (ANOVA) test were used for comparison between two or more than two groups, respectively. Reported p-values were multiplicity adjusted using a Tukey test. Analysis of Covariance (ANCOVA) was used to compare linear regression slopes. Differences were considered statistically significant for p < 0.05, with the following thresholds: *p < 0.05, **p < 0.01, ***p < 0.001.Supplementary Information