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In this chapter, we’ll go through the basic building blocks of web pages such as HTML and CSS and demonstrate scraping structured information from them using popular Python libraries such as Beautiful Soup and lxml. Later, we’ll expand our knowledge and tackle issues that will make our scraper into a full-featured web crawler capable of fetching information from multiple web pages.

In this chapter, we’ll talk about an open source dataset called common crawl which is available on AWS’s registry of open data (https://registry.opendata.aws/).

In this chapter, we will discuss a crawling framework called Scrapy and go through the steps necessary to crawl and upload the web crawl data to an S3 bucket.

In this chapter, you will learn the fundamentals of cloud computing and get an overview of select products from Amazon Web Services. AWS offers a free tier where a new user can access many of the services free for a year, and this will make almost all examples here close to free for you to try out. Our goal is that by the end of this chapter, you will be comfortable enough with AWS to perform almost all the analysis in the rest of the book on the AWS cloud itself instead of locally.

In this chapter, we’ll learn about processing web crawl data on a big data scale using distributed computing architecture using Amazon Web Services (AWS).

In the preceding chapters, we have solely relied on the structure of the HTML documents themselves to scrape information from them, and that is a powerful method to extract information.

Appetite is tightly linked to the sensory experience of feeding, including the smell, taste, and sight of food. Sensory perception can affect the palatability of food, modulating appetite beyond homeostatic requirements. Hypothalamic neurons that govern feeding are responsive to sensory cues associated with food, including food odors. However, the circuit mechanisms by which sensory information is processed and relayed to feeding nodes to affect feeding behavior is not well understood. Recent work has identified a population of excitatory basal forebrain neurons that modulate potent appetite suppression, as well as respond to food-associated and innately aversive odorants. To investigate this circuitry, we stereotaxically targeted virus expressing Cre-dependent channelrhodopsin to the basal forebrain and implanted fiber optic cannulas over the injection site. Mice were allowed to recover and underwent training to form a passive association of chow with a unique monomolecular odorant. After training, mice were fasted overnight, and were then presented with both the food-associated odor as well as a similar, novel odor in zones of an arena with and without photostimulation. To evaluate whether stimulation of this circuitry influenced sensory modulation of feeding behavior, video recording and behavioral tracking analysis were used to compare time spent investigating either odor. Thus, this protocol provides a useful paradigm to assay the contribution of different circuits in appetitive and aversive behaviors.

The field of articular cartilage repair has made significant advances in recent decades; yet current therapies are generally not evaluated or tested, at the time of pivotal trial, in patients with a variety of common comorbidities. To that end, we systematically reviewed cartilage repair clinical trials to identify common exclusion criteria and reviewed the literature to identify emerging regenerative approaches that are poised to overcome these current exclusion criteria. The term “knee cartilage repair” was searched on clinicaltrials.gov. Of the 60 trials identified on initial search, 33 were further examined to extract exclusion criteria. Criteria excluded by more than half of the trials were identified in order to focus discussion on emerging regenerative strategies that might address these concerns. These criteria included age (55 years old), small defects (2), large defects (>8 cm2), multiple defect (>2 lesions), BMI >35, meniscectomy (>50%), bilateral knee pathology, ligamentous instability, arthritis, malalignment, prior repair, kissing lesions, neurologic disease of lower extremities, inflammation, infection, endocrine or metabolic disease, drug or alcohol abuse, pregnancy, and history of cancer. Finally, we describe emerging tissue engineering and regenerative approaches that might foster cartilage repair in these challenging environments. The identified criteria exclude a majority of the affected population from treatment, and thus greater focus must be placed on these emerging cartilage regeneration techniques to treat patients with the challenging “red knee”.

Appetite is driven by nutritional state, environmental cues, mood, and reward pathways. Environmental cues strongly influence feeding behavior, as they can dramatically induce or diminish the drive to consume food despite homeostatic state. Here, we have uncovered an excitatory neuronal population in the basal forebrain that is activated by food-odor related stimuli, and potently drives hypophagia. Notably, we found that the basal forebrain directly integrates environmental sensory cues to govern feeding behavior, and that basal forebrain signaling, mediated through projections to the lateral hypothalamus, promotes selective avoidance of food and food-related stimuli. Together, these findings reveal a novel role for the excitatory basal forebrain in regulating appetite suppression through food avoidance mechanisms, highlighting a key function for this structure as a potent integrator of sensory information towards governing consummatory behaviors.

The menisci are fibrocartilaginous tissues that are crucial to the load-sharing and stability of the knee, and when injured, these properties are compromised. Meniscus replacement scaffolds have utilized the circumferential alignment of fibers to recapitulate the microstructure of the native meniscus; however, specific consideration of size, shape, and morphology has been largely overlooked. The purpose of this study was to personalize the fiber-reinforcement network of a meniscus reconstruction scaffold. Human cadaveric menisci were measured for a host of tissue (length, width) and subtissue (regional widths, root locations) properties, which all showed considerable variability between donors. Next, the asymmetrical fiber network was optimized to minimize the error between the dimensions of measured menisci and predicted fiber networks, providing a 51.0% decrease (p = 0.0091) in root-mean-square (RMS) error. Finally, a separate set of human cadaveric knees was obtained, and donor-specific fiber-reinforced scaffolds were fabricated. Under cyclic loading for load-distribution analysis, in situ implantation of personalized scaffolds following total meniscectomy restored contact area (253.0 mm2 to 488.9 mm2, p = 0.0060) and decreased contact stress (1.96 MPa to 1.03 MPa, p = 0.0025) to near-native values (597.4 mm2 and 0.83 MPa). Clinical use of personalized meniscus devices that restore physiologic contact stress distributions may prevent the development of post-traumatic osteoarthritis following meniscal injury.

Cartilage injuries and subsequent tissue deterioration impact millions of patients. Since the regeneration of functional hyaline cartilage remains elusive, methods to stabilize the remaining tissue, and prevent further deterioration, would be of significant clinical utility and prolong joint function. Finite element modeling shows that fortification of the degenerate cartilage (Reinforcement) and reestablishment of a superficial zone (Sealing) are both required to restore fluid pressurization within the tissue and restrict fluid flow and matrix loss from the defect surface. Here, a hyaluronic acid (HA) hydrogel system is designed to both interdigitate with and promote the sealing of the degenerated cartilage. Interdigitating fortification restores both bulk and local pericellular tissue mechanics, reestablishing the homeostatic mechanotransduction of endogenous chondrocytes within the tissue. This HA therapy is further functionalized to present chemo mechanical cues that improve the attachment and direct the response of mesenchymal stem/stromal cells at the defect site, guiding localized extracellular matrix deposition to "seal" the defect. Together, these results support the therapeutic potential, across cell and tissue length scales, of an innovative hydrogel therapy for the treatment of damaged cartilage.

Atypical food intake is a primary cause of obesity and other eating and metabolic disorders. Insight into the neural control of feeding has previously focused mainly on signalling mechanisms associated with the hypothalamus1-5, the major centre in the brain that regulates body weight homeostasis6,7. However, roles of non-canonical central nervous system signalling mechanisms in regulating feeding behaviour have been largely uncharacterized. Acetylcholine has long been proposed to influence feeding8-10 owing in part to the functional similarity between acetylcholine and nicotine, a known appetite suppressant. Nicotine is an exogenous agonist for acetylcholine receptors, suggesting that endogenous cholinergic signalling may play a part in normal physiological regulation of feeding. However, it remains unclear how cholinergic neurons in the brain regulate food intake. Here we report that cholinergic neurons of the mouse basal forebrain potently influence food intake and body weight. Impairment of cholinergic signalling increases food intake and results in severe obesity, whereas enhanced cholinergic signalling decreases food consumption. We found that cholinergic circuits modulate appetite suppression on downstream targets in the hypothalamus. Together our data reveal the cholinergic basal forebrain as a major modulatory centre underlying feeding behaviour.

Our objective was to test the efficacy of collagen-hyaluronan scaffolds reinforced with poly(l-lactic acid) (PLLA) fibers in an ovine total meniscus replacement model. Scaffolds were implanted into 9 sheep (n = 1 at 8 weeks, n = 2 at 16 weeks, n = 3 at both 24, 32 weeks) following total medial meniscectomy. From 16 weeks on, explants were characterized by confined compression creep, histological, and biochemical analyses. Articular surfaces were observed macroscopically and damage was ranked histologically using the Mankin score. At sacrifice, three of the nine PLLA scaffolds had completely ruptured, and the intact scaffolds experienced progressive shape changes and severe narrowing in the body region at 16, 24, and 32 weeks. Aggregate compressive modulus and permeability did not improve with time. Histological and biochemical analyses showed significantly less extracellular matrix and less matrix organization compared to native tissue. Osteophytes, bone erosion, and cartilage damage were observed, increasing with time postimplantation. A buildup of lactic acid and/or the rapid loss of scaffold mechanical integrity due to PLLA degradation are probable causes for the joint abnormalities observed in this study. These results are in sharp contrast to those of our previous successful total meniscus replacement studies using polyarylate [p(DTD DD)] fiber-reinforced scaffolds. This suggests that PLLA fiber as produced in this study cannot be used as reinforcement for a meniscus replacement scaffold.

This article focuses on the routes of transport and abiotic processes involved in the environmental transformation of synthetic organic chemicals and how molecular structure controls the products and lifetimes of several important classes of organic chemicals. The chapter also discusses the current methods to reliably determine the rates and products of degradation of new chemicals based on combinations of chemical structure and environmental processes as well as use of laboratory and field measurements. Methods are also discussed for use of structure activity relations for this purpose.

Collagen-based scaffolds are extensively used in biomaterials and tissue engineering applications. These scaffolds have shown great biocompatibility and versatility, but their relatively low mechanical properties may limit use in orthopaedic load-bearing applications. Moreover, terminal sterilization with gamma irradiation, as is commonly performed with commercial devices, presents concerns over structural integrity and enzymatic stability. Therefore, the goal of this study was to test the hypothesis that EDC/NHS cross-linking (10 mM/5 mM) can protect collagen-hyaluronan sponges from the damaging effects of gamma irradiation. Specifically, we evaluated compressive and tensile mechanical properties, enzymatic stability, porosity and pore size, and swelling ratio. Ultimate tensile strength and elastic modulus exhibited increases (168.5 and 245.8%, respectively) following irradiation, and exhibited over tenfold increases (1049.2 and 1270.6%, respectively) following cross-linking. Irradiation affected pore size (38.4% decrease), but cross-linking prior to irradiation resulted in only a 17.8% decrease. Cross-linking also showed an offsetting effect on the equilibrium modulus, enzymatic stability, and swelling ratio of sponges. These results suggest that carbodiimide cross-linking of collagen-hyaluronan sponges can mitigate the structural damage typically experienced during gamma irradiation, warranting their use in tissue engineering applications.

Inhibitory interneurons are integral to sensory processing, yet revealing their cell type-specific roles in sensory circuits remains an ongoing focus. To Investigate the mouse olfactory system, we selectively remove GABAergic transmission from a subset of olfactory bulb interneurons, EPL interneurons (EPL-INs), and assay odor responses from their downstream synaptic partners — tufted cells and mitral cells. Using a combination of in vivo electrophysiological and imaging analyses, we find that inactivating this single node of inhibition leads to differential effects in magnitude, reliability, tuning width, and temporal dynamics between the two principal neurons. Furthermore, tufted and not mitral cell responses to odor mixtures become more linearly predictable without EPL-IN inhibition. Our data suggest that olfactory bulb interneurons, through exerting distinct inhibitory functions onto their different synaptic partners, play a significant role in the processing of odor information., The precise cell-type specific role of inhibitory interneurons in regulating sensory responses in the olfactory bulb is not known. Here, the authors report that removing GABAergic inhibition from one layer differentially affects response dynamics of the two main output cell types and changes odor mixture processing.

Background: Patients who are diagnosed with heart failure (HF) are almost universally characterized by left ventricular ejection fraction (LVEF) at the time of diagnosis, where the most common dichotomy is preserved LVEF (HFpEF, LVEF > 50%) or reduced LVEF (HFrEF, LVEF < 40%). The concept of recovered LVEF (< 40% to > =50%) is considered by some to indicate a separate category of HF. We sought to study the natural history of a patient’s LVEF using natural language processing among a large cohort of patients. Methods: We identified 457,457 patients in the VA Health Care System between 2005 and 2016 with a first outpatient diagnosis of heart failure with an LVEF between 6 months before or one year after the heart failure diagnosis and a second LVEF after the diagnosis. We used all LVEF data from 2005-2016 obtained from previously validated natural language processing of echo reports. LVEF at baseline was grouped into deciles (1-29, 30-39, 40-49, 50-59, 60-69 and >=70%). Results: The 457,457 patients had 8,857,436 echos over a mean 4 +/- 3 years of follow-up (median 11 echos per patient). At the time of diagnosis the mean age was 69 +/- 11 years, 97.6% male, 19% were African-American, 45% had a diagnosis of diabetes, and 61% had a diagnosis of ischemic heart disease. During follow-up among those with an initial LVEF < 40%, between 21% and 31% recovered their LVEF to over 50% at some point during follow-up (Table). A similar frequency was noted for those dropping to an LVEF < 40% if the initial LVEF was high. When the last LVEF was used to define change, then a recovered LVEF (>50%) was less common (9% if initial LVEF was < 20%, 8% if the initial LVEF was 20-29%, 13% if the initial LVEF was 30-39%). The final LVEF was < 40% in 30% of patients with an initial LVEF of 40-49%, 15% if the initial LVEF was 50-59%, 9% if the initial LVEF was 60-69%, and 6% if the initial LVEF was >=70%. Conclusion: In this multiyear study of a national cohort, LVEF varied substantially over time, with a recovered LVEF (=50%) noted at some point in 20-30% depending on initial LVEF. However, when the last LVEF is used, recovery occurred in only 8-13%. Thus, approximately half of patients with a recovered LVEF at some point will revert to depressed LVEF.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2–4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Background:Meniscus injuries in the United States result in an estimated 850,000 surgical procedures each year. Although meniscectomies are the most commonly performed orthopaedic surgery, little advancement has been made in meniscus replacement and regeneration, and there is currently no total meniscus replacement device approved by the Food and Drug Administration.Hypothesis:A novel fiber-reinforced meniscus scaffold can be used as a functional total meniscus replacement.Study Design:Controlled laboratory study.Methods:A tyrosine-derived, polymer fiber–reinforced collagen sponge meniscus scaffold was evaluated mechanically (tensile and compressive testing) and histologically after 16 and 32 weeks of implantation in an ovine total meniscectomy model (N = 20; 16 implants plus 4 meniscectomies, divided equally over the 2 time periods). The extent of cartilage damage was also measured on tibial plateaus by use of toluidine blue surface staining and on femoral condyles by use of Mankin scores on histological slides.Results:Scaffolds induced formation of neomeniscus tissue that remained intact and functional, with breaking loads approximating 250 N at both 16 and 32 weeks compared with 552 N for native menisci. Tensile stiffness values (99 and 74 N/mm at 16 and 32 weeks, respectively) were also comparable with those of the native meniscus (147 N/mm). The compressive modulus of the neomeniscus tissue (0.33 MPa at both 16 and 32 weeks) was significantly increased compared with unimplanted (time 0) scaffolds (0.15 MPa). There was histological evidence of extensive tissue ingrowth and extracellular matrix deposition, with immunohistochemical evidence of types I and II collagen. Based on significantly decreased surface damage scores as well as Mankin scores, the scaffold implants provided greater protection of articular cartilage compared with the untreated total meniscectomy.Conclusion:This novel fiber-reinforced meniscus scaffold can act as a functional meniscus replacement, with mechanical properties similar to those of the native meniscus, while protecting the articular cartilage of the knee from the extensive damage after a total meniscectomy.Clinical Relevance:This meniscus replacement scaffold has the potential to improve surgical treatment and provide better long-term outcomes for those suffering from severe meniscus damage.

Wnt signaling plays an essential role in developmental and regenerative myelination of the CNS, therefore it is critical to understand how the factors associated with the various regulatory layers of this complex pathway contribute to these processes. Recently, Apcdd1 was identified as a negative regulator of proximal Wnt signaling, however its role in oligodendrocyte (OL) differentiation and reymelination in the CNS remain undefined. Analysis of Apcdd1 expression revealed dynamic expression during OL development, where its expression is upregulated during differentiation. Functional studies using ex vivo and in vitro OL systems revealed that Apcdd1 promotes OL differentiation, suppresses Wnt signaling, and associates with β-catenin. Application of these findings to white matter injury (WMI) models revealed that Apcdd1 similarly promotes OL differentiation after gliotoxic injury in vivo and acute hypoxia ex vivo. Examination of Apcdd1 expression in white matter lesions from neonatal WMI and adult multiple sclerosis revealed its expression in subsets of oligodendrocyte (OL) precursors. These studies describe, for the first time, the role of Apcdd1 in OLs after WMI and reveal that negative regulators of the proximal Wnt pathway can influence regenerative myelination, suggesting a new therapeutic strategy for modulating Wnt signaling and stimulating repair after WMI.

Controlled racemization of enantiopure alcohols is a key step in dynamic kinetic resolution. We recently reported the racemization of enantiopure phenyl-ring-containing alcohols using W110A Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (W110A TeSADH), which relies on selectivity mistakes. Trp-110 lines the large pocket of the active site of TeSADH, which allows W110A TeSADH mutant to accommodate phenyl-ring-containing substrates in Prelog mode, albeit with selectivity mistakes. Here, we report the racemization of enantiopure phenyl-ring-containing alcohols using several Trp-110 mutants of TeSADH in the presence of the oxidized and reduced forms of nicotinamide–adenine dinucleotide. We observed a noticeable enhancement in racemization efficiency when W110G TeSADH was used compared with W110Q, W110M, W110L, W110I, and W110V. This observation was anticipated because the W110G mutation is expected to open the large pocket of the active site to a greater extent compared to other mutants of TeSADH at W110. Both enantiomers of 1-phenyl-2-propanol and 4-phenyl-2-butanol were fully racemized by W110G TeSADH. We also constructed a triple mutant of TeSADH, W110A/I86A/C295A, by site-directed mutagenesis with the aim of opening the two pockets of the active site of TeSADH. The W110A/I86A/C295A mutant was employed to racemize enantiopure phenyl-ring-containing alcohols. The current study demonstrates that W110G and W110A/I86A/C295A TeSADH are more efficient catalysts for the racemization of enantiopure secondary alcohols than the previously reported mutant W110A TeSADH [6] .

The ability to control the substrate specificity and stereochemistry of enzymatic reactions is of increasing interest in biocatalysis. As this review highlights, this control can be achieved throug...

Meniscus injuries represent one of the most-common intra-articular knee injuries. The current treatment options include meniscectomy and allograft transplantation, both with poor long-term outcomes. Therefore, there is a need for regenerative techniques to restore meniscal function. To preclinically test scaffolds for meniscus replacement, large animal models need to be established and standardized. This review establishes the anatomical and compositional similarities between human and sheep menisci and provides guidance for implantation and evaluation of such devices. The ovine meniscus represents a scaled-down version of the human meniscus, with only slight structural differences that can be addressed during device fabrication. Implantation protocols in sheep remain a challenge, as the meniscus cannot be visualized with the arthroscopic-assisted procedures commonly performed in human patients. Thus, we recommend the appropriate implantation protocols for meniscus visualization, ligamentous restoration, and surgical fixation of both total and partial meniscus replacement devices. Last, due to the lack of standardization in evaluation techniques, we recommend a comprehensive battery of tests to evaluate the efficacy of meniscus replacement implants. We recommend other investigators utilize these surgical and testing techniques to establish the ovine model as the gold standard for preclinical evaluation of meniscus replacement devices.

Cheminformatics-based software tools can predict the molecular structure of transformation products using a library of transformation reaction schemes. This paper presents the development of such a library for abiotic hydrolysis of organic chemicals under environmentally relevant conditions. The hydrolysis reaction schemes in the library encode the process science gathered from peer-reviewed literature and regulatory reports. Each scheme has been ranked on a scale of one to six based on the median half-life in a data set compiled from literature-reported hydrolysis rates. These ranks are used to predict the most likely transformation route when more than one structural fragment susceptible to hydrolysis is present in a molecule of interest. Separate rank assignments are established for pH 5, 7, and 9 to represent standard conditions in hydrolysis studies required for registration of pesticides in Organisation for Economic Co-operation and Development (OECD) member countries. The library is applied to predict the likely hydrolytic transformation products for two lists of chemicals, one representative of chemicals used in commerce and the other specific to pesticides, to evaluate which hydrolysis reaction pathways are most likely to be relevant for organic chemicals found in the natural environment.

Background: Numerous quality metrics for heart failure care now exist based on process (from clinical guidelines) or outcome (readmission and mortality). It is unclear, however, how valid these measures are at serving as indicators of hospital quality for patients with heart failure (HF). We sought to determine validity by comparing convergent validity (correlation among measures) and reliability (correlation over time). Methods: We gathered data on several HF performance measures from the Veterans Affairs Health Care System from 2008-2013. Process data are collected through chart review of a random sample of facilities. There are between 114 and 119 facilities that have at least one patient that is a treatment candidate each year. There are a mean 72 patients per facility per year included. For each facility and each year, we determined, among ideal candidates, use of ACE-inhibitor (ACEi), any beta-blocker (BB), recommended beta-blocker (rBB) (carvedilol, metoprolol succinate, bisoprolol), mineralocorticoid receptor antagonist (MRA), hydralazine/isosorbide dinitrate in African Americans, as well as rates of 30-day readmission, 30-day mortality, and 1-year mortality. We measured validity in 2 ways: determining correlations between measures, assuming that if a measure is associated with overall “quality” that it should be correlated with the other measures, and year-to-year reliability, assuming that if a measure has a good signal/noise ratio then there should be a strong correlation from one year to the next. Results: Data were available for 55,735 hospitalizations for HF from 2008-2013. In determining correlations between performance measures, we found a statistically significant correlation between ACEi and rBB (R=0.25, p Conclusion: BB, ACEi and MRA use rates showed evidence of validity as measures of HF quality through correlation with each other, mortality and/or year to year reliability. In contrast, 30-day readmission was poorly correlated with process of care and mortality and had poor reliability.

The authors present an image-rich case-based review of common endovascular renal interventions, with emphasis on technical aspects and teaching points.

Alcohol dehydrogenases (ADHs) are enzymes that catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. We have been studying a thermostable, nicotinamide-adenine dinucleotide phosphate (NADP+)-dependent, secondary ADH from Thermoanaerobacter ethanolicus (TeSADH). In the current work, we expanded our library of TeSADH and adopted the site-saturation mutagenesis approach in creating a comprehensive mutant library at W110. We used phenylacetone as a model substrate to study the effectiveness of our library because this substrate showed low enantioselectivity in our previous work when reduced using W110A TeSADH. Five of the newly designed W110 mutants reduced phenylacetone at >99.9% ee, and two of these mutants exhibit an enantiomeric ratio (E-value) of over 100. These five mutants also reduced 1-phenyl-2-butanone and 4-phenyl-2-butanone to their corresponding (S)-configured alcohols in >99.9% ee. These new mutants of TeSADH will likely have synthetic utility for reduction of aromatic ketones in the future.

Memory access rate is a primary performance bottleneck in high-performance networking systems. The MoSys Bandwidth Engine family of integrated circuits provides a significant improvement in effective memory performance by using high-speed serial I/O's, many banks of memory, a low-latency, highly efficient protocol, and intelligence within the device. The first member of the family can perform 2 billion 72-bit reads per second or 1 billion read-modify-write operations per second.

The skeletal system provides structure, protection, and movement to the body through bones, cartilages, tendons, and ligaments. Many congenital, traumatic, and degenerative diseases may affect the function of skeletal tissues during the life span, leading to the necessity of very specific replacements and treatments. In the widespread and mechanically constraining scenario of skeletal pathologies, biodegradable polymers can play unique roles that should not only be confined to adjuvant bulk devices. Tissue engineering has recently renewed the attention towards this class of biomaterials, enchantingly exploiting their outstanding versatility to accomplish smart and biomimetic solutions to surgical and therapeutic needs. This chapter describes the most recent achievements in this field, focusing on tissue type- and subtype-specific replacements, while taking into account clinical applications and future trends.

Background:Meniscus injuries and resulting meniscectomies lead to joint deterioration, causing pain, discomfort, and instability. Tissue-engineered devices to replace the meniscus have not shown consistent success with regard to function, mechanical integrity, or protection of cartilage.Purpose:To evaluate a novel resorbable polymer fiber–reinforced meniscus reconstruction scaffold in an ovine model for 52 weeks and assess its integrity, tensile and compressive mechanics, cell phenotypes, matrix organization and content, and protection of the articular cartilage surfaces.Study Design:Controlled laboratory study.Methods:Eight skeletally mature ewes were implanted with the fiber-reinforced scaffold after total meniscectomy, and 2 additional animals had untreated total meniscectomies. Animals were sacrificed at 52 weeks, and the explants and articular surfaces were analyzed macroscopically. Explants were characterized by ultimate tensile testing, confined compression creep testing, and biochemical, histological, and immunohistochemical analyses. Cartilage damage was characterized using the Mankin score on histologic slides from both the femur and tibia.Results:One sheep was removed from the study because of a torn extensor tendon; the remaining 7 explants remained fully intact and incorporated into the bone tunnels. All explants exhibited functional tensile loads, tensile stiffnesses, and compressive moduli. Fibrocartilagenous repair with both types 1 and 2 collagen were observed, with areas of matrix organization and biochemical content similar to native tissue. Narrowing in the body region was observed in 5 of 7 explants. Mankin scores showed less cartilage damage in the explant group (femoral condyle: 3.43 ± 0.79, tibial plateau: 3.50 ± 1.63) than in the meniscectomy group (femoral condyle: 8.50 ± 3.54, tibial plateau: 6.75 ± 2.47) and were comparable with Mankin scores at the previously reported 16- and 32-week time points.Conclusion:A resorbable fiber-reinforced meniscus scaffold supports formation of functional neomeniscus tissue, with the potential to prevent joint degeneration that typically occurs after total meniscectomy. Further studies with improvements to the initial mechanics of the scaffold and testing for longer time periods are warranted.Clinical Relevance:Meniscectomy is an extremely common orthopaedic procedure, and few options currently exist for the treatment of significant loss of meniscus tissue. Successful development of a tissue-engineered meniscus scaffold could substantially reduce the incidence of postmeniscectomy joint degeneration and the subsequent procedures used for its treatment.

Trypanosoma cruzi was demonstrated in blood smears and heart tissue from a 5-year old, female, English Cocker Spaniel that had never been outside of the state of Virginia, USA. Plasma from the dog was positive in a commercially available immunochromatographic dipstick assay for T. cruzi and negative in an immunochromatographic dipstick assay for visceral Leishmania spp. The plasma from the dog had an indirect immunofluorescent antibody titer of 1:800 against epimastigotes of T. cruzi while the titer was 1:50 against promastigotes of L. infantum. The parasite was isolated from the blood in vitro from the dog (TcVT-1 isolate) and used to experimentally infect female C3H and ICR mice. The parasite was nonpathogenic for experimentally inoculated mice. DNA was isolated from parasites grown in vitro and used to determine that the genotype of T. cruzi present in the dog was genotype TcIV. This genotype is common in raccoons, Procyon lotor, in North America and suggests that raccoons may serve as reservoirs for canine infection.

OBJECTIVES/SPECIFIC AIMS: Focal cartilage injuries of the knee joint are common and present a treatment challenge due to minimal intrinsic repair. Cartilage tissue engineering techniques currently used in clinical practice are expensive, cumbersome, and often ineffective in patients with mechanical or medical comorbidities. To address these issues, we developed an acellular nanofibrous scaffold with encapsulated growth factors designed to enhanced articular cartilage repair. Our goal is to evaluate this technology in vitro and pilot a large animal model for eventual translation into human subjects. METHODS/STUDY POPULATION: Hyaluronic acid (HA, 65 kDa) will be methacrylated (~40% modification, MeHA) and conjugated with cell-adhesive (RGD) groups. A solution of 4% wt/vol MeHA, 2% wt/vol polyethylene oxide (900 kDa), 0.05% wt/vol Irgacure 2959, and 0.005% wt/vol stromal cell-derived factor-1α (SDF-1α) and/or transforming growth factor-β3 (TGF-β3) will be prepared in ddH2O. The solution will be electrospun onto a rotating mandrel to achieve a dry scaffold thickness of 0.5 mm. The scaffold matt will be UV cross-linked and 5 mm-diameter samples will be cut out. Four groups of scaffolds will be prepared: MeHA, MeHA+SDF, MeHA+TGF, MeHA+SDF+TGF. All groups will be evaluated for fiber diameter, swell thickness, equilibrium compressive modulus, degradation rate, and growth factor release rate over 4 weeks (n=10). Scaffolds will also be seeded with juvenile porcine MSCs (5×104) in 200 μL of medium incubated for 24 hours. Seeded scaffolds will be evaluated for equilibrium compressive modulus, cell infiltration, and chondrogenesis at 4 and 8 weeks (n=10). Scaffolds will then be evaluated in a juvenile Yucatan minipig cartilage defect model. In total, 6 animals will undergo bilateral knee surgery to create four 4 mm-diameter full-thickness cartilage defects in each trochlear grove. All defects will receive microfracture to release marrow elements. Each knee will receive 2 scaffolds of the same group (replicates) with paired microfracture controls, resulting in a sample size of 3. Animals will be sacrificed at 12 weeks and defects will be evaluated via non-destructive indentation testing for mechanical properties, microCT for defect fill and subchondral bone morphology, and histology for ICRS II Visual Histological Assessment Scoring. RESULTS/ANTICIPATED RESULTS: Our preliminary studies have shown reliable replication of electrospun MeHA scaffolds. We anticipate cross-linking density to correlate positively with compressive modulus, and negatively with swell thickness, degradation rate, and growth factor release rate. We anticipate the addition of SDF-1α and TGF-β3 to increase cell infiltration and chondrogenesis, respectively, within seeded scaffolds. Similarly, we expect minipig defects treated with growth factor-releasing scaffolds to show greater mechanical properties, defect fill, and ICRS II score compared with MeHA scaffolds without growth factor. DISCUSSION/SIGNIFICANCE OF IMPACT: This study has the potential to show how an HA-based cell-free scaffold can be augmented with 2 growth factors that act synergistically to improve cartilage repair in a large animal model. This technology would improve upon the cell-free scaffolds already used clinically for autologous matrix-induced chondrogenesis and is directly translatable.

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and this drug leads to decreased levels of low density lipoprotein (LDL) cholesterol. Lower LDL cholesterol has direct relationship in reducing mortality from coronary heart diseases. Lipitor® (atorvastatin calcium) was the first drug to reach the annual sales of 10 billion dollars in USA and currently is the top selling pharmaceutical product globally. Atorvastatin has a side chain containing two chiral centers as its pharmacophore and it can be synthesized either from chiral pool precursors, by using metal catalysts; or more preferably by the application of free or immobilized enzymes and whole cell biocatalysts for carrying out either asymmetric synthesis or racemic resolution. Biocatalytic synthesis methods for chiral atorvastatin intermediates employ a wide variety of biocatalysts such as alcohol dehydrogenase, 2-deoxy- d -ribose 5-phosphate aldolase, nitrilase, lipase, etc. and each of these biocatalytic processes is discussed in detail in this paper.

Summary The α-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"—the hinge that mediates opening and closing of the RNAP active center cleft—to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related α-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.

The effects of hydrostatic pressure and temperature on the reaction of secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus with enantiomers of 2-butanol, 2-pentanol, and 2-hexanol have been measured. For all substrates, increasing pressure favors the S enantiomer, whereas increasing temperature favors the R enantiomer. Fitting of the pressure and temperature data for 2-hexanol provided apparent ΔΔS‡ and ΔΔV‡ values of +46 ± 15 J/mol and +(2.0 ± 0.4) × 10–2 L/mol, respectively. These results support our previous proposal that desolvation of the enzyme active site plays an important role in stereospecificity.

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset, dominantly inherited neurodegenerative disease caused by expansion of a glutamine repeat tract in ataxin-1 (ATXN1). Although the precise function of ATXN1 remains elusive, it seems to be involved in transcriptional repression. We find that mutant ATXN1 represses transcription of the neurotrophic and angiogenic factor vascular endothelial growth factor (VEGF). Genetic overexpression or pharmacologic infusion of recombinant VEGF mitigates SCA1 pathogenesis, suggesting a new therapeutic strategy for this disease.

Wnt signaling plays an essential role in developmental and regenerative myelination of the CNS, therefore it is critical to understand how the factors associated with the various regulatory layers of this complex pathway contribute to these processes. Recently, Apcdd1 was identified as a negative regulator of proximal Wnt signaling, however its role in oligodendrocyte (OL) differentiation and reymelination in the CNS remain undefined. Analysis of Apcdd1 expression revealed dynamic expression during OL development, where its expression is upregulated during differentiation. Functional studies using ex vivo and in vitro OL systems revealed that Apcdd1 promotes OL differentiation, suppresses Wnt signaling, and associates with β-catenin. Application of these findings to white matter injury (WMI) models revealed that Apcdd1 similarly promotes OL differentiation after gliotoxic injury in vivo and acute hypoxia ex vivo. Examination of Apcdd1 expression in white matter lesions from neonatal WMI and adult multiple sclerosis revealed its expression in subsets of oligodendrocyte (OL) precursors. These studies describe, for the first time, the role of Apcdd1 in OLs after WMI and reveal that negative regulators of the proximal Wnt pathway can influence regenerative myelination, suggesting a new therapeutic strategy for modulating Wnt signaling and stimulating repair after WMI.

Objectives: Arthroscopic partial meniscectomy is one of the most commonly performed orthopaedic procedures. Although the procedure provides good symptom relief, long term follow up suggests the procedure results in an early onset of degenerative knee arthritis in a significant percentage of patients. Currently, treatment options for lost meniscal tissue are extremely limited and those available do not provide a long term solution. Therefore, there exists a need for a functional meniscus replacement in order to prevent joint deterioration. The objective of this project was to test a cross-linked collagen-hyaluronan sponge reinforced with synthetic, resorbable poly(DTD DD) fibers for meniscal implantation in an ovine model. Methods: Meniscus scaffolds were fabricated from poly(DTD DD) fibers woven into a semi-lunar wedge shape with extended tails for rigid tibial fixation. A dispersion of hyaluronic acid and type I bovine collagen was injected into the woven fiber scaffold. The scaffold was then lyophilized, crosslinked, and irradiated. The time-zero mechanical properties of the scaffold were evaluated with ultimate tensile testing and compression creep testing, and for load sharing function with a novel hoop stress evaluation and joint pressure distribution using Tekscan monitoring. The scaffolds were evaluated in an in vivo ovine model. A total medial meniscectomy was performed in the right hind leg of 30 sheep. Twenty-four of these sheep received a tissue engineered scaffold. The scaffold was anchored to the tibial plateau at the anterior and posterior root locations with titanium interference screws and sutured to the medial capsule. The remaining 6 sheep did not receive an implant and served as controls. Eight experimental and two control sheep were sacrificed at 16, 32 and 52 weeks. Scaffolds and adjacent articular cartilage underwent comprehensive mechanical and histological evaluation. Results: Pre-implantation characterization: Ultimate tensile strength of the implant was 660 N. The compressive modulus was 0.15 MPa. Hoop stress evaluation demonstrated a linear correlation between joint axial load and tensile stress in the implant. Tekscan evaluation demonstrated the implant increased joint contact area and decreased peak contact stress. In vivo evaluation demonstrated, at all time points, all 24 implants were fully intact and well healed to the surrounding capsule and maintained the meniscus-like shape (Figure 1). Gross and histological evaluation of the articular cartilage adjacent to the implant demonstrated minimal degenerative change in experimental knees. Control knees demonstrated advanced cartilage degradation adjacent to the meniscal resection. Robust tissue ingrowth into the implants was histologically demonstrated with tissue deposition occurring in a pattern consistent with tensile stresses in the implant. The tensile strength of the scaffold explant was 255 N at 16 weeks and 237 N at 32 weeks and 210 N at 52 weeks. The compressive modulus was 0.29 MPa at 16 weeks, 0.34 MPa at 32 weeks, and 0.49 MPa at 52 weeks. Conclusion: The results of this study support the feasibility of a tissue engineered load sharing scaffold for treatment of significant meniscal damage. The scaffold has the potential to prevent degenerative changes that occur after meniscectomy. Longer term studies will be necessary to confirm the true chondroprotective capabilities of this scaffold.

Heart disease is a leading cause of death worldwide, and coronary heart disease causes 1 of every 6 deaths in the United States [1]. Following a myocardial infarction, scar tissue gradually replaces myocardium that is lost through a process of collagen deposition and an increase in tensile strength of the tissue [2]. This leads to infarct expansion, adverse ventricular remodeling and dysfunction, and ultimately heart failure. Dilation of the left ventricle (LV) leads to increased LV wall stress and is ultimately responsible for adverse ventricular remodeling. LV dilation causes stretching and thereby increased wall stress, prohibiting cardiomyocytes from effectively contracting, which leads to further dilation, and ultimately a decrease in cardiac pump efficiency [3]. Previously, it has been shown that using a tissue filler to modify the material properties of an infarct limits the process of ventricular remodeling [4]. Angiogenesis is another mechanism by which adverse ventricular remodeling can be limited. Previously, our group developed engineered stromal cell-derived factor-1α (ESA), a computationally designed analog of an established endothelial progenitor cell chemokine, SDF-1α, and demonstrated that ESA injection enhances LV function by promoting angiogenesis and retains the native properties of the extracellular matrix (ECM) [5] [6]. In this study, we propose that injection of ESA to infarcted cardiac muscle improves the tensile strength and viscoelastic properties of ventricular muscle.Copyright © 2013 by ASME

The mechanical force-induced activation of the adhesive protein von Willebrand Factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (pVWF) multimers to bind platelets. Here we showed that a pathological level of high shear stress exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers.

METHODS: 103 patients with degenerative mitral regurgitation (MR) underwent single-suture repair of the mitral valve through a 3-cm right chest incision from May 2007 through December 2012. The prolapsed segment of the mitral valve was imbricated with a double running CV 5 Gore-Tex suture, resulting in a smooth plane of coaptation. Preoperative and perioperative echocardiograms as well as patient outcomes were analyzed and compared to patients undergoing minimally invasive isolated mitral valve repair using quadrangular resection at the same institution during the same time period.

The effect of electrode porosity on the electrochemical response of redox active molecules (potassium ferricyanide, ruthenium(III) hexammine, and ferrocene methanol) in the presence of bovine serum albumin or fibrinogen was studied at macroporous (pore diameter: 1200 nm), hierarchical (1200/60 nm), and nanoporous (