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DNA is damaged through various exogenous sources (e.g., automobile exhaust, tobacco smoke, and processed foods), which can yield diverse C8-dG bulky aryl adducts. Adducts are known to induce structural changes to DNA that can lead to various biological outcomes, ranging from cell death to diseases such as cancer. Unfortunately, the relationship between the chemical composition of the damaged product, the adducted DNA structure, and the biological consequences is not well understood, which limits the development of disease detection and prevention strategies. The present study uses density functional theory (DFT) calculations and quintuplicate 1 μs molecular dynamics (MD) simulations to characterize the structure of DNA containing 21 model C8-dG adducts that systematically differ in size (phenyl to pyrenyl), shape (α (2,3), β (3,4) fusion, or ring substitution), and nucleobase–aryl group linkage (N, O, and C-linked). DFT calculations reveal that the inherent structural features of the G nucleobase adducts are impacted by linker type and bulky moiety shape, but not size, with the conformational flexibility reducing with α-ring fusion and linker composition as N > O > C. These structural properties are maintained in nucleoside models, which also reveal an increased propensity for anti-to-synrotation about the glycosidic bond with N < O < C linker type. Although these diverse chemical features do not influence the global structure of adducted DNA, the adducts differentially impact the conformation local to the adducted site, including the relative populations of structures with the bulky moiety in the major groove (B conformer) and intercalated (stacked) into the helix (S conformer). Specifically, while the smallest phenyl adducts favor the B conformation and the largest pyrenyl-derived adducts stabilize the S conformation, the B/S ratio decreases with an increase in ring size and N > O > C linker composition. The shape and size (length) of the adduct can further finetune the B/S ratio, with β-fused naphthyl or α-fused phenanthryl N-linked adducts and O or C-linked adducts containing ring substitution increasing the prevalence of the S adducted DNA conformation. Overall, this work uncovers the significant effect of bulky moiety size and linker type, as well as the lesser impact of aryl group shape, on adducted DNA structure, which suggests differential replication and repair outcomes, and thereby represents an important step toward rationalizing connections between the structure and biological consequences of diverse DNA adducts.

Purpose Persistent velopharyngeal insufficiency (VPI) following primary palatoplasty remains a difficult problem to treat. This study evaluates speech outcomes following revision palatoplasty with tissue augmentation using buccal myomucosal flaps (BMF) as an alternative to pharyngoplasty for patients with VPI.Methods A retrospective single-center review of revision palatoplasty with tissue augmentation at a tertiary pediatric hospital Cleft-Craniofacial Center between January 2017 and March 2021 was conducted. Patients with a history of previous palatoplasty, a diagnosis of persistent or recurrent VPI, and comprehensive pre- and postoperative speech evaluations who underwent revision palatoplasty with BMF were included.Results Twenty patients met inclusion criteria (35% female, 20% syndromic). Mean age at the time of revision palatoplasty with BMF was 9.7 years. Preoperatively, all patients had stigmatizing speech and received the recommendation for speech surgery; the mean Pittsburgh Weighted Speech Score (PWSS) was 14.3 ± 4.9. The mean postoperative PWSS at the most recent assessment was 4.2 ± 2.3, representing a statistically significant improvement from preoperative scores (P < .001). Mean follow-up time was 8.9 months. Following revision palatoplasty with BMF, only one patient has received the recommendation for further speech surgery. No complications were noted.Conclusion In patients with VPI following primary palatoplasty, revision palatoplasty with tissue augmentation offers an alternative to pharyngoplasty. This approach preserves dynamic velopharyngeal function, improves speech outcomes, and should be considered an option when treating patients with post-primary palatoplasty VPI.

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Supplemental Digital Content is available in the text

ABSTRACTThis article reports on professional development of nine U.S. social work students of color who participated in a delegation exploring human trafficking in Thailand through a reverse mission program. Through the use of the 2015 Council on Social Work Education’s (CSWE) Educational Policy and Accreditation Standards (EPAS) as a conceptual framework, this qualitative study provides insight into delegates’ development related to professional roles and identities, understanding the complexity of social contexts, enhancing cultural humility, and sparking community engagement at home in the United States. This study points to the importance of critical reflection and peer support as tools to inspire delegates to take direct action upon return to their own contexts. Discussion of findings and implications for social work educators and students are included.

ABSTRACTAn intricate network of internal plastid membrane is formed in chloroplasts during plant ontogenesis. The network develops directly from a simple membrane system in proplastids or through a paracrystalline tubular prolamellar body (PLB) in etioplasts. A complex spatial structure of PLB interconnected with porous prothylakoids (PTs) is formed when the seedling growth proceeds without light. Upon illumination, PLB transforms through a multistage process into a lamellar system of stacked (grana) and loosely arranged (stroma) thylakoids. The chloroplast thylakoid network, the site of photochemical reactions, is one of the most important as well as most complicated membrane systems in nature. Thus, extensive studies on its possible spatial rearrangements are important for a better understanding of the crucial role of the thylakoid structure in the regulation of photosynthesis. Complementary structural methods are necessary to provide a complex image of spatial thylakoid arrangement. Therefore, in this review, we describe the results obtained by methods using both fixed and in vivosamples. The main goal of this review is to present the state of research on the three-dimensional (3D) structure of internal plastid membrane network, its spatial arrangement during plant ontogenesis and its modifications caused by different factors. Influence of different membrane components on the formation of the thylakoid network shape is also described. Additionally, we summarize recent 3D structural results in the field of thylakoid dynamics induced by environmental factors, especially of light-triggered changes related to rearrangements of the photosynthetic complexes.

Objective: To evaluate postoperative pain, hospital length of stay (LOS), and associated costs of multiple perioperative analgesic strategies following alveolar bone grafting (ABG).Design: Retrospective comparative cohort study.Setting: Tertiary care pediatric hospital.Patients/Participants: Iliac crest bone graft (ICBG) harvest techniques: “Open Harvest” (n = 22), “Trephine Only” (n = 14), or “Trephine + Pain Pump” (n = 25).Intervention: The “Open Harvest” group underwent open ICBG harvest with 3-walled osteotomies. For the other 2 treatment groups, a trephine drill was used to harvest iliac crest bone with a ropivacaine infusion pump into the hip donor site (“Trephine + Pain Pump”) or without (“Trephine Only”). Patients who underwent ABG with only cadaveric allograft were analyzed as a comparison group (“No Harvest”).Main Outcomes Measures: Outcomes were planned prior to data collection: maximum pain score, hospital LOS, and associated health care costs.Results: Maximum pain scores were significantly higher in the “Open Harvest” group (7.3/10) compared to “Trephine + Pain Pump” (1.8/10; P< .0001) and “No Harvest” groups (2.8/10; P< .01). Hospital LOS decreased from 2.4 days (“Open Harvest”) to 0.5 days (Trephine + Pain Pump”; P< .0001). Twelve (48%) patients from “Trephine + Pain Pump” were discharged on the day of surgery. The “Trephine + Pain Pump” saved an estimated $5336 for a unilateral ABG and $7265 for a bilateral ABG compared to “Open Harvest.”Conclusions: The combined use of the trephine ICBG technique and ropivacaine infusion catheter effectively decreased pain, shortened hospital stay, and improved cost saving compared to patients who have undergone other methods of ICBG.

Historically, the approach to pediatric cranioplasty has been largely extrapolated from the treatment of adults. More recently, however, the intricacies of pediatric cranial reconstruction have become better understood, and the surgical management has been refined contemporaneously. Each patient’s cranial defect bears a unique set of challenges and, as such, the choice of cranioplasty technique must be tailored accordingly.

Synovial chondromatosis is a rare, benign condition characterized by metaplasia of the synovial membrane with formation of multiple cartilaginous nodules within the synovium. This case is of a 58-year-old male who initially presented with right knee pain. The patient was ultimately found to have extensive synovial chondromatosis. The diagnosis was confirmed by histopathologic evaluation and treated with loose body removal and radical synovectomy via an arthroscopic approach.

The effect of alkaline-earth-metal alkoxides on the protonolysis of Cp2M′Cl2(M′ = Ti, Zr, Hf; Cp = cyclopentadiene) was investigated. This approach enabled the design of compounds with well-defined molecular structures to generate high-purity binary metal oxides. Single-source molecular precursors with structures of [M2M′2(μ3-OEt)2(μ-OEt)4(OEt)6(EtOH)4] with M = Mg and M′ = Ti (1), Zr (2), and Hf (3), [Ca6Ti4(μ6-O)2(μ4-O)2(μ3-OEt)12(OEt)12(EtOH)6Cl4] (4), and [M2M′2(μ4-O)(μ-OEt)5(OEt)4(EtOH)4Cl]nwith M = Ca and M′ = Zr (5) and Hf (6) were prepared via elimination of the cyclopentadienyl ring from Cp2M′Cl2as CpH in the presence of M(OEt)2and ethanol (EtOH) as a source of protons. Meanwhile, similar reactions involving the initial substitution of Cl ligands by OEt groups in Cp2M′Cl2(M′ = Ti, Zr, Hf) resulted in the formation of [M2M′2(μ3-OEt)2(μ-OEt)4(OEt)6(EtOH)4] with M = Ca and M′ = Ti (7), Zr (8), and Hf (9). The precursors were characterized by elemental analysis, NMR spectroscopy, and single-crystal X-ray structural analysis. Magnesium compounds 1–3decomposed at 750–850 °C to give MgTiO3along with small amounts of Mg2TiO4, Mg2Zr5O12, or Mg2Hf5O12binary metal oxides. The thermolysis of calcium compounds 4and 7–9led to highly pure CaTiO3, CaZrO3, or CaHfO3perovskite-like oxide particles with diameters of 20–30 nm.

Background:As Comprehensive Stroke Centers (CSCs) strive to improve neurointerventional (NIR) times, process improvements have been put in place to streamline workflows. Our prior publication (VISIION) demonstrated an improvement in key performance indicators (KPIs) in our CSC. The purpose of this study is to analyze whether the positive results demonstrated were sustainable.Methods:Sequential stroke NIR patients being Direct Arriving LVO (DALVO) and telemedicine transfer LVO (BEMI) cases were assessed, including subgroups of DALVO-OnHours, DALVO-OffHours, BEMI-OnHours, and BEMI-OffHours. We analyzed times for the original 6 months pre (6/10/20-1/15/21) and compared them to a 17 months post-implementation (1/16/21- 6/25/22) to evaluate for sustainability. Mann-Whitney U was utilized.Results:150 NIR cases were analyzed pre (n=47) v. post (n=103) Viz.ai implementation (DALVO-OnHours 7 v. 20, DALVO-OffHours 10 v. 25, BEMI-OnHours 13 v. 20, BEMI-OffHours 17 v. 38). For Door-to-groin (DTG) assessments, improvement was noted for DALVO-OffHours 39% (157min,96min;p<0.001), DALVO-ALL 25% (127min,95min;p=0.006), BEMI-OffHours 46% (45min,25min;p=0.023), and BEMI-ALL 40% (42min,25min;p=0.005). Activation-to-groin (ATG), door-to-device (DTD), and door-to-recanalization (DTR) showed similar improvements. For DALVO-OffHours, there were significant reductions in door to CT (DTC) 81% (26min,5min;p<0.001), ATG 32% (90min,61min;p=0.036), DTG 39% (157min,96min;p<0.001), DTD 31% (178min,123min;p=0.002), and DTR 32% (197min,135min;p=0.003).Conclusions:Consistent with our initial 6 month post-implementation pilot, we noted sustainability over a 17 month period with sustained reduction in KPIs for numerous key NIR subgroups. In the greatest opportunity subgroup (DALVO-OffHours), requiring team mobilization off hours without benefit of telemedicine transfer lead time, we noted a significant reduction in all 5 time metrics. Our sustainability finding is important to show that process improvements continued even after the immediate period, making a Hawthorne effect less likely and adding credibility to the results. Models such as this, could be useful for other centers striving to optimize workflow and improve NIR times.

ABSTRACTFollowing analysis of primary cervix, vagina, and first-void female urine specimens for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalisvia commercial transcription-mediated amplification (TMA), residual material was subjected to Mycoplasma genitaliumresearch-use-only TMA. Representation within a 2,478-specimen retrospective study set was established by comparison to a 6-month audit of clinical C. trachomatisTMA (12,999 specimens) on the basis of the C. trachomatisdetection rate, specimen source distribution, clinic location, and age. M. genitaliumwas detected in 282 (11.4%) patients. This rate was higher than those seen with T. vaginalis(9.0%; P= 0.005), C. trachomatis(6.2%), and N. gonorrhoeae(1.4%). Positive M. genitaliumresults were confirmed by repeat testing or alternative-target TMA at a rate of 98.7%. The mean age of the M. genitalium-infected females (24.7 years) was lower than that of the T. vaginalis-infected females (mean, 30.1 years; P< 0.0001) and higher than that of the C. trachomatis-infected females (mean, 23.8 years; P= 0.003). Of 566 patient encounters positive for at least one sexually transmitted infection (STI), 35.9% exhibited sole detection of M. genitalium(P= 0.0004 versus sole detection of other STI agents) and 26.1% were solely positive for T. vaginalis(P< 0.0002 versus C. trachomatis). The M. genitaliumand T. vaginalisdetection rates among 755 patients at urban emergency departments were 14.6% and 13.0%, respectively (P= 0.37). A 10.0% M. genitaliumdetection rate from other facilities exceeded that of T. vaginalis(7.2%; P= 0.004). Incorporation of M. genitaliumTMA into comprehensive testing programs would detect M. genitaliumin a significant proportion of females, particularly those in outpatient obstetrics and gynecology (OB/GYN) settings.

Background Despite decades of craniofacial surgeons repairing cleft palates, there is no consensus for the rate of fistula formation following surgery. The authors present a meta-analysis of studies that reported on primary cleft palate to determine the rate of oronasal fistula and to identify risk factors for their development.Methods A literature search for the period between 2000 and 2012 was performed. Articles were queried and strict inclusion and exclusion criteria were applied to focus on primary cleft palate repair. A meta-analysis of these data was conducted.Results The meta-analysis included 11 studies, comprising 2505 children. The rate of oronasal fistula development was 4.9% (95% confidence interval, 3.8% to 6.1%). When analyzing a larger cohort, there was a significant relationship between Veau classification and the occurrence of a fistula (P< .001), with fistulae most prevalent in patients with a Veau IV cleft. The most common location for a fistula was at the soft palate-hard palate junction. One study used decellularized dermis in cleft repair with a fistula rate of 3.2%.Conclusions Using 11 studies comprising 2505 children, we find the rate of reported fistula occurrence to be 4.9%. Furthermore, patients with a Veau IV cleft are significantly more likely to develop an oronasal fistula. When fistulae do occur, they do so most often at the soft palate-hard palate junction. A deeper understanding of fistula formation will help cleft palate surgeons improve their outcomes in the operating room and will allow them to effectively communicate expectations with patients' families in the clinic.

The article presents information on the "Golden Age" of 17th-century Dutch still life paintings from 1602 to 1702. The depiction of musical instruments, food, and flowers in Dutch still lives is examined, the collecting of Dutch still life paintings is discussed, and information is offered on the works of Dutch painters including Gerrit Dou, Carel van Vogelaer, and Cornelis Norbertus Gysbrechts. Differences in prices of sales at aution of still life paintings featuring various themes are compared.

We review the anatomy of the jugular fossa in the context of current computed tomography and magnetic resonance techniques with examples of common and unusual presentations of these masses. Contrast-enhanced computed tomography and magnetic resonance imaging both can suggest etiology; define the size and full intracranial, transforaminal, and extracranial extent; and evaluate the regional anatomy to optimize surgical approach and minimize cranial nerve deficits. As inadequate exposure is a leading contributor to incomplete resection and recurrence, the ability to reformat high-resolution imaging in multiple planes is an asset to surgical planning. [Copyright &y& Elsevier]

Although the microanatomy of the membranous labyrinth remains beyond the capacity of clinical scanners, developments in imaging technology and postprocessing have continued to improve resolution of subtle intralabyrinthine pathology. This article reviews imaging selection and optimization for labyrinthine symptoms, limitations owing to patient and technical factors, and examples of discrete pathology and inferred findings based on imaging characteristics. In addition to the desire to improve resolution and contrast within a clinically acceptable acquisition time, current imaging dilemmas include computed tomography dose reduction strategies and optimizing imaging utilization. [Copyright &y& Elsevier]

This study aimed to evaluate the relationship between cochlear signal on fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI) sequences and hearing in patients undergoing hearing preservation surgery for vestibular schwannoma (VS) and to demonstrate a new classification system to be used in imaging evaluation of patients with VS.

Management of high-risk lesions of the breast can be challenging because of confusing terminology and changes in management options. Likewise, the imaging identification of high-risk lesions of the breast may be challenging because of overlap with benign and malignant appearances. Despite the recurrent controversies in the management of high-risk lesions of the breast, it is imperative to understand the proper classification and management options of such lesions. This knowledge will help the radiologist communicate confidently with both the referring physician and the patient. The imaging appearances by mammogram, ultrasound, and dynamic contrast-enhanced breast MRI are illustrated for various high-risk lesions of the breast. A management algorithm is provided as a reference for the clinical recommendations at the time of diagnosis of high-risk lesions of the breast.

Optimal management of pediatric mandible fractures demands that the practitioner balance reduction and fixation with preservation of growth potential and function. The ideal synthesis of these goals has not yet been defined. The authors catalogue their experience with pediatric mandible fractures at a major pediatric teaching hospital with reference to demographics, injury type, treatment, and outcomes to inform future management of these injuries.

Be independent: Under basic conditions a furan ring entrapped in an aromatic macrocycle readily recovered the properties characteristic for a free molecule and assimilated a water molecule forming the Achmatowicz rearrangement product with a 3‐pyranone subunit built in. Coordination of palladium(II) created a complex stabilizing the second tautomer with two sp3carbon atoms (oxygen red, water green, palladium purple, and the aryl group blue).

Unabhängig: Im Basischen zeigte ein Furanring, der in einem aromatischen Makrozyklus eingeschlossen war, die Eigenschaften eines freien Moleküls und nahm ein Wassermolekül auf, wodurch das Achmatowicz‐Umlagerungsprodukt mit einer 3‐Pyranon‐Einheit entstand. Palladium(II)‐Koordination ergab einen Komplex, der ein zweites Tautomer durch zwei sp3‐C‐Atome stabilisierte (Sauerstoff rot, Wasser grün, Palladium violett und die Aryl‐Gruppe blau).

Pediatric craniofacial fractures are anatomically distinct from their adult counterparts and must be managed with respect for future growth and development. These injuries must be approached as entities fundamentally different from adult craniofacial fractures. Here, the authors aim to provide context for practitioners managing pediatric facial fractures by augmenting presently available demographic, diagnostic, and treatment data.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered on an absorbable collagen sponge is a U.S. Food and Drug Administration-approved therapy shown to be an effective means of generating bone formation in multiple clinical settings. However, the optimum dose and delivery of rhBMP-2 to the calvaria are undetermined. The aim of the authors' study was to investigate the use of rhBMP-2 in addressing calvarial defects in a large-animal model through a variety of modifications to this U.S. Food and Drug Administration-approved therapy.

Wireless Sensor Networks (WSNs) are gaining an increasing industry wide adoption. However, there remain major challenges such as network dimensioning and node placement especially in Built Environment Networks (BENs). Decisions on the node placement, orientation, and the number of nodes to cover the area of interest are usually ad-hoc. Ray tracing tools are traditionally employed to predict RF signal propagation; however, such tools are primarily intended for outdoor environments. RF signal propagation varies greatly indoors due to building materials and infrastructure, obstacles, node placement, antenna orientation and human presence. Because of the complexity of signal prediction, these factors are usually ignored or given little weight when such networks are analyzed. The paper’s results show the effects of the building size and layout, building materials, human presence and mobility on the signal propagation of a BEN. Additionally, they show that antenna radiation pattern is a key factor in the RF propagation performance, and appropriate device orientation and placement can improve the network reliability. Further, the RSS facility in RF transceivers can be exploited to detect the presence and motion of humans in the environment.

Reports have identified cells capable of osteogenic differentiation in bone marrow, muscle, and adipose tissues, but there are few direct comparisons of these different cell types. Also, few have investigated the potential connection between a tissue-specific abnormality and cells derived from seemingly unrelated tissues. In this article, the authors compare cells isolated from wild-type rabbits or rabbits with nonsyndromic craniosynostosis, defined as the premature fusion of one or more of the cranial sutures.

Response Evaluation Criteria in Solid Tumors Version 1.1 RECIST 1.1 has added positron emission tomography PET as an optional complement for the detection of new lesions. In this study, we evaluate the utility of fluorodeoxyglucose FDG-PET in the identification of new lesions and progressive disease not recognized on computed tomography CT in patients with nonsmall cell lung cancer NSCLC undergoing therapy.

The purpose of this study was to demonstrate spatial control of osteoblast differentiation in vitroand bone formation in vivousing inkjet bioprinting technology and to create three-dimensional persistent bio-ink patterns of bone morphogenetic protein-2 (BMP-2) and its modifiers immobilized within microporous scaffolds. Semicircular patterns of BMP-2 were printed within circular DermaMatrix™ human allograft scaffold constructs. The contralateral halves of the constructs were unprinted or printed with BMP-2 modifiers, including the BMP-2 inhibitor, noggin. Printed bio-ink pattern retention was validated using fluorescent or 125I-labeled bio-inks. Mouse C2C12 progenitor cells cultured on patterned constructs differentiated in a dose-dependent fashion toward an osteoblastic fate in register to BMP-2 patterns. The fidelity of spatial restriction of osteoblastic differentiation at the boundary between neighboring BMP-2 and noggin patterns improved in comparison with patterns without noggin. Acellular DermaMatrix constructs similarly patterned with BMP-2 and noggin were then implanted into a mouse calvarial defect model. Patterns of bone formation in vivowere comparable with patterned responses of osteoblastic differentiation in vitro. These results demonstrate that three-dimensional biopatterning of a growth factor and growth factor modifier within a construct can direct cell differentiation in vitroand tissue formation in vivoin register to printed patterns.

ABSTRACTBorrelia burgdorferi, the Lyme disease-causing spirochete, can persistently infect its vertebrate hosts for years. B. burgdorferiis often found associated with host connective tissue, where it interacts with components of the extracellular matrix, including fibronectin. Some years ago, a borrelial surface protein, named BBK32, was identified as a fibronectin-binding protein. However, B. burgdorferiBBK32 mutants are still able to bind fibronectin, indicating that the spirochete possesses additional mechanisms for adherence to fibronectin. We now demonstrate that RevA, an unrelated B. burgdorferiouter surface protein, binds mammalian fibronectin in a saturable manner. Site-directed mutagenesis studies identified the amino terminus of the RevA protein as being required for adhesion to fibronectin. RevA bound to the amino-terminal region of fibronectin. RevA binding to fibronectin was not inhibited by salt or heparin, suggesting that adhesin-ligand interactions are primarily nonionic and occur through the non-heparin-binding regions of the fibronectin amino-terminal domains. revAgenes are widely distributed among Lyme disease spirochetes, and the present studies determined that all RevA alleles tested bound fibronectin. In addition, RevB, a paralogous protein found in a subset of B. burgdorferistrains, also bound fibronectin. We also confirmed that RevA is produced during mammalian infection but not during colonization of vector ticks and determined that revAtranscription is controlled through a mechanism distinct from that of BBK32.

Borrelia burgdorferi, the Lyme disease-causing spirochete, can persistently infect its vertebrate hosts for years. B. burgdorferi is often found associated with host connective tissue, where it interacts with components of the extracellular matrix, including fibronectin. Some years ago, a borrelial surface protein, named BBK32, was identified as a fibronectin-binding protein. However, B. burgdorferi BBK32 mutants are still able to bind fibronectin, indicating that the spirochete possesses additional mechanisms for adherence to fibronectin. We now demonstrate that RevA, an unrelated B. burgdorferi outer surface protein, binds mammalian fibronectin in a saturable manner. Site-directed mutagenesis studies identified the amino terminus of the RevA protein as being required for adhesion to fibronectin. RevA bound to the amino-terminal region of fibronectin. RevA binding to fibronectin was not inhibited by salt or heparin, suggesting that adhesin-ligand interactions are primarily nonionic and occur through the non-heparin-binding regions of the fibronectin amino-terminal domains. revA genes are widely distributed among Lyme disease spirochetes, and the present studies determined that all RevA alleles tested bound fibronectin. In addition, RevB, a paralogous protein found in a subset of B. burgdorferi strains, also bound fibronectin. We also confirmed that RevA is produced during mammalian infection but not during colonization of vector ticks and determined that revA transcription is controlled through a mechanism distinct from that of BBK32.

This chapter describes common laboratory procedures that can reduce the risk of culture contaminations (sepsis), collectively referred as “aseptic technique.” Two major strategies of aseptic work are described: using a Bunsen burner and a laminar flow hood. Both methods are presented in the form of general protocols applicable to a variety of laboratory tasks such as pipetting and dispensing aliquots, preparing growth media, and inoculating, passaging, and spreading microorganisms on petri dishes. Curr. Protoc. Microbiol. 11:A.4D.1‐A.4D.11. © 2008 by John Wiley & Sons, Inc.

Peptidylglycine α-amidating monooxygenase (PAM) converts inactive terminal-glycine prohormones into their activated α-amidated forms. PAM is thought to play a role in the development of antiandrogen drug resistance in prostate cancer (CaP) through PAM-activated autocrine growth. On the basis of the previous finding that many lung cancer cell lines excrete PAM into their culture media, this study investigates PAM levels in media collected from human CaP cell line cultures. Androgen-independent DU145 and PC-3 prostate cancer cell lines exhibited readily detectable levels of PAM activity in extracts and media, whereas the androgen-dependent LNCaP cell line showed little or no activity. Because of the much larger volume of media versus cell extracts, more than 90% of the total PAM activity was located in the media for both the PC-3 and DU145 cell lines, providing a readily accessible source of CaP PAM. A simple, scalable method to obtain PAM from the culture media of androgen-independent human prostate cancer cell lines is described in this article. This approach provides a much easier means of collecting CaP-derived PAM than previously described cell fractionation procedures and should facilitate the investigations of the role and targeting of PAM in hormone-independent CaP.

ABSTRACTSerological diagnosis of Lyme disease may be complicated by antigenic differences between infecting organisms and those used as test references. Accordingly, it would be helpful to include antigens whose sequences are well conserved by a broad range of Lyme disease spirochetes. In the present study, line blot analyses were performed using recombinant complement regulator-acquiring surface protein 2 (BbCRASP-2) from Borrelia burgdorferisensu stricto strain B31 and serum samples from human Lyme disease patients from throughout the United States and Germany. The results indicated that a large proportion of the patients had produced antibodies recognizing recombinant BbCRASP-2. In addition, Lyme disease spirochetes isolated from across North America and Europe were found to contain genes encoding proteins with high degrees of similarity to the B. burgdorferitype strain B31 BbCRASP-2, consistent with the high percentage of serologically positive patients. These data indicate that BbCRASP-2 may be valuable for use in a widely effective serological assay.

Serological diagnosis of Lyme disease may be complicated by antigenic differences between infecting organisms and those used as test references. Accordingly, it would be helpful to include antigens whose sequences are well conserved by a broad range of Lyme disease spirochetes. In the present study, line blot analyses were performed using recombinant complement regulator-acquiring surface protein 2 (BbCRASP-2) from Borrelia burgdorferi sensu stricto strain B31 and serum samples from human Lyme disease patients from throughout the United States and Germany. The results indicated that a large proportion of the patients had produced antibodies recognizing recombinant BbCRASP-2. In addition, Lyme disease spirochetes isolated from across North America and Europe were found to contain genes encoding proteins with high degrees of similarity to the B. burgdorferi type strain B31 BbCRASP-2, consistent with the high percentage of serologically positive patients. These data indicate that BbCRASP-2 may be valuable for use in a widely effective serological assay.

The Lyme disease spirochete, Borrelia burgdorferi, is largely resistant to being killed by its hosts’ alternative complement activation pathway. One possible resistance mechanism of these bacteria is to coat their surfaces with host complement regulators, such as factor H. Five different B. burgdorferi outer surface proteins having affinities for factor H have been identified: complement regulator-acquiring surface protein 1 (BbCRASP-1), encoded by cspA; BbCRASP-2, encoded by cspZ; and three closely related proteins, BbCRASP-3, -4, and -5, encoded by erpP, erpC, and erpA, respectively. We now present analyses of the recently identified BbCRASP-2 and cspZ expression patterns throughout the B. burgdorferi infectious cycle, plus novel analyses of BbCRASP-1 and erp-encoded BbCRASPs. Our results, combined with data from earlier studies, indicate that BbCRASP-2 is produced primarily during established mammalian infection, while BbCRASP-1 is produced during tick-to-mammal and mammal-to-tick transmission stages but not during established mammalian infection, and Erp-BbCRASPs are produced from the time of transmission from infected ticks into mammals until they are later acquired by other feeding ticks. Transcription of cspZ and synthesis of BbCRASP-2 were severely repressed during cultivation in laboratory medium relative to mRNA levels observed during mammalian infection, and cspZ expression was influenced by culture temperature and pH, observations which will assist identification of the mechanisms employed by B. burgdorferi to control expression of this borrelial infection-associated protein.

ABSTRACTThe Lyme disease spirochete, Borrelia burgdorferi, is largely resistant to being killed by its hosts’ alternative complement activation pathway. One possible resistance mechanism of these bacteria is to coat their surfaces with host complement regulators, such as factor H. Five different B. burgdorferiouter surface proteins having affinities for factor H have been identified: complement regulator-acquiring surface protein 1 (BbCRASP-1), encoded by cspA; BbCRASP-2, encoded by cspZ; and three closely related proteins, BbCRASP-3, -4, and -5, encoded by erpP, erpC, and erpA, respectively. We now present analyses of the recently identified BbCRASP-2 and cspZexpression patterns throughout the B. burgdorferiinfectious cycle, plus novel analyses of BbCRASP-1 and erp-encoded BbCRASPs. Our results, combined with data from earlier studies, indicate that BbCRASP-2 is produced primarily during established mammalian infection, while BbCRASP-1 is produced during tick-to-mammal and mammal-to-tick transmission stages but not during established mammalian infection, and Erp-BbCRASPs are produced from the time of transmission from infected ticks into mammals until they are later acquired by other feeding ticks. Transcription of cspZand synthesis of BbCRASP-2 were severely repressed during cultivation in laboratory medium relative to mRNA levels observed during mammalian infection, and cspZexpression was influenced by culture temperature and pH, observations which will assist identification of the mechanisms employed by B. burgdorferito control expression of this borrelial infection-associated protein.

The causative agent of Lyme disease, Borrelia burgdorferi, is naturally resistant to its host's alternative pathway of complement-mediated killing. Several different borrelial outer surface proteins have been identified as being able to bind host factor H, a regulator of the alternative pathway, leading to a hypothesis that such binding is important for borrelial resistance to complement. To test this hypothesis, the development of B. burgdorferi infection was compared between factor H-deficient and wild-type mice. Factor B- and C3-deficient mice were also studied to determine the relative roles of the alternative and classical/lectin pathways in B. burgdorferi survival during mammalian infection. While it was predicted that B. burgdorferi should be impaired in its ability to infect factor H-deficient animals, quantitative analyses of bacterial loads indicated that those mice were infected at levels similar to those of wild-type and factor B- and C3-deficient mice. Ticks fed on infected factor H-deficient or wild-type mice all acquired similar numbers of bacteria. Indirect immunofluorescence analysis of B. burgdorferi acquired by feeding ticks from the blood of infected mice indicated that none of the bacteria had detectable levels of factor H on their outer surfaces, even though such bacteria express high levels of surface proteins capable of binding factor H. These findings demonstrate that the acquisition of host factor H is not essential for mammalian infection by B. burgdorferi and indicate that additional mechanisms are employed by the Lyme disease spirochete to evade complement-mediated killing.

ABSTRACTThe causative agent of Lyme disease, Borrelia burgdorferi, is naturally resistant to its host's alternative pathway of complement-mediated killing. Several different borrelial outer surface proteins have been identified as being able to bind host factor H, a regulator of the alternative pathway, leading to a hypothesis that such binding is important for borrelial resistance to complement. To test this hypothesis, the development of B. burgdorferiinfection was compared between factor H-deficient and wild-type mice. Factor B- and C3-deficient mice were also studied to determine the relative roles of the alternative and classical/lectin pathways in B. burgdorferisurvival during mammalian infection. While it was predicted that B. burgdorferishould be impaired in its ability to infect factor H-deficient animals, quantitative analyses of bacterial loads indicated that those mice were infected at levels similar to those of wild-type and factor B- and C3-deficient mice. Ticks fed on infected factor H-deficient or wild-type mice all acquired similar numbers of bacteria. Indirect immunofluorescence analysis of B. burgdorferiacquired by feeding ticks from the blood of infected mice indicated that none of the bacteria had detectable levels of factor H on their outer surfaces, even though such bacteria express high levels of surface proteins capable of binding factor H. These findings demonstrate that the acquisition of host factor H is not essential for mammalian infection by B. burgdorferiand indicate that additional mechanisms are employed by the Lyme disease spirochete to evade complement-mediated killing.

The possibility that non-sedating antihistamines could elicit sedation in mice due to drug-induced inhibition of brain PgP was evaluated by measuring the ability of desloratadine alone or in combination with verapamil to cause ataxia in mice. Also, the concentrations of desloratadine in plasma and in brain homogenates were measured by liquid chromatography- mass spectrometry. Relative to methylcellulose (control) treatment, verapamil plus desloratadine decreased rotarod performance of mice. Plasma concentrations of desloratadine appeared comparable in the mice treated with either desloratadine or verapamil plus desloratadine, however the rate of decline of desloratadine from brain tissue was slower in mice treated with verapamil plus desloratadine compared to mice treated with desloratadine only. These data suggest that inhibition of brain PgP can convert desloratadine to a sedating antihistamine in mice.

We investigated the intra- and interrater reliability of ischemic lesion volumes measurements assessed by different MRI sequences at various times from onset.