Tuning through-space interactions via the secondary coordination sphere of an artificial metalloenzyme leads to enhanced Rh(iii)-catalysis.

We report computationally-guided protein engineering of monomeric streptavidin Rh(iii) artificial metalloenzyme to enhance catalysis of the enantioselective coupling of acrylamide hydroxamate esters and styrenes. Increased TON correlates with calculated distances between the Rh(iii) metal and surrounding residues, underscoring an artificial metalloenzyme's propensity for additional control in metal-catalyzed transformations by through-space interactions.

Disparities between Antibody Occupancy, Orientation, and Cytotoxicity in Immunotherapy.

We report fusion proteins designed to bind spatially distinct epitopes on the extracellular portion of HER2, a breast cancer biomarker and established therapeutic target, and recruit IgG (either anti-His6 or serum IgG) to the cell surface. When the proteins were incubated with anti-His6 antibody and various concentrations of a single HER2-binding protein His6 fusion, we observed interference and a decrease in antibody recruitment at HER2-binding protein concentrations exceeding ∼30 nM. In contrast, concomitant treatment with two or three distinct HER2-binding protein His6 fusions, and anti-His6 , results in increased antibody recruitment, even at relatively high HER2-binding protein concentration. In some instances, increased antibody recruitment leads to increased antibody-dependent cellular cytotoxicity (ADCC) activity. While a fusion protein consisting of a HER2-binding nanobody and Sac7d, a protein evolved to recognize the Fc domain of IgG, binds IgG from serum, antibody recruitment does not lead to ADCC activity. Rationales for these disparities are provided. Collectively, our findings have implications for the design of efficacious targeted immunotherapeutic biologics, and ensembles thereof.

Asymmetric δ-Lactam Synthesis with a Monomeric Streptavidin Artificial Metalloenzyme.

Reliable design of artificial metalloenzymes (ArMs) to access transformations not observed in nature remains a long-standing and important challenge. We report that a monomeric streptavidin (mSav) Rh(III) ArM permits asymmetric synthesis of α,ß-unsaturated-δ-lactams via a tandem C-H activation and [4+2] annulation reaction. These products are readily derivatized to enantioenriched piperidines, the most common N-heterocycle found in FDA approved pharmaceuticals. Desired δ-lactams are achieved in yields as high as 99% and enantiomeric excess of 97% under aqueous conditions at room temperature. Embedding a Rh cyclopentadienyl (Cp*) catalyst in the active site of mSav results in improved stereocontrol and a 7-fold enhancement in reactivity relative to the isolated biotinylated Rh(III) cofactor. In addition, mSav-Rh outperforms its well-established tetrameric forms, displaying 11-33 times more reactivity.

Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation.

PURPOSE: Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts. PROCEDURES: sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [18F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [18F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[18F]RL-I conjugates. RESULTS: [18F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97-100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [125I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95-99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [18F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively, and was similar to that seen for [125I]SGMIB-2Rs15d. Uptake of [18F]RL-I-2Rs15d in subcutaneous xenografts was 16-20 %ID/g over 1-3 h. Subcutaneous tumor could be clearly delineated by microPET/CT imaging with [18F]RL-I-2Rs15d but not with [18F]RL-I-R3B23. Intracranial breast cancer brain metastases could be visualized after intravenous administration of both [18F]RL-I-2Rs15d and [18F]RL-I-5F7. CONCLUSIONS: Although radiolabeled 2Rs15d conjugates exhibited lower tumor cell retention both in vitro and in vivo than that observed previously for 5F7, given that it binds to a different epitope on HER2 from those targeted by the clinically utilized HER2-targeted therapeutic antibodies trastuzumab and pertuzumab, F-18-labeled 2Rs15d has potential for assessing HER2 status by PET imaging after trastuzumab and/or pertuzumab therapy.

Minimalist Antibodies and Mimetics: An Update and Recent Applications.

The immune system utilizes antibodies to recognize foreign or disease-relevant receptors, initiating an immune response to destroy unwelcomed guests. Because researchers can evolve antibodies to bind virtually any target, it is perhaps unsurprising that these reagents, and their small-molecule conjugates, are used extensively in clinical and basic research environments. However, virtues of antibodies are countered by significant challenges. Foremost among these is the need for expression in mammalian cells (largely due to often necessary post-translational modifications). In response to these challenges, researchers have developed an array of minimalist antibodies and mimetics, which are smaller, more stable, simpler to express in Escherichia coli, and amendable to laboratory evolution and protein engineering. Here we describe these scaffolds and discuss recent applications of minimalist antibodies and mimetics.

Patients' administration preferences: progesterone vaginal insert (Endometrin®) compared to intramuscular progesterone for Luteal phase support.

BACKGROUND: Administration of exogenous progesterone for luteal phase support has become a standard of practice. Intramuscular (IM) injections of progesterone in oil (PIO) and vaginal administration of progesterone are the primary routes of administration. This report describes the administration preferences expressed by women with infertility that were given progesterone vaginal insert (PVI) or progesterone in oil injections (PIO) for luteal phase support during fresh IVF cycles. METHODS: A questionnaire to assess the tolerability, convenience, and ease of administration of PVI and PIO given for luteal phase support was completed by infertile women diagnosed with PCOS and planning to undergo IVF. The women participated in an open-label study of highly purified human menopausal gonadotropins (HP-hMG) compared with recombinant FSH (rFSH) given for stimulation of ovulation. RESULTS: Most women commented on the convenience and ease of administration of PVI, while a majority of women who administered IM PIO described experiencing pain. In addition, their partners often indicated that they had experienced at least some anxiety regarding the administration of PIO. The most distinguishing difference between PVI and PIO in this study was the overall patient preference for PVI. Despite the need to administer PVI either twice a day or three times a day, 82.6% of the patients in the PVI group found it "very" or "somewhat convenient" compared with 44.9% of women in the PIO group. CONCLUSIONS: The results of this comprehensive, prospective patient survey, along with findings from other similar reports, suggest that PVI provides an easy-to-use and convenient method for providing the necessary luteal phase support for IVF cycles without the pain and inconvenience of daily IM PIO. Moreover, ongoing pregnancy rates with the well-tolerated PVI were as good as the pregnancy rates with PIO. TRIAL REGISTRATION: ClinicalTrial.gov, NCT00805935.

State-mandated insurance coverage is associated with the approach to hydrosalpinges before IVF.

The aim of this study was to determine whether practice in states with infertility insurance mandates is associated with physician-reported practice patterns regarding hydrosalpinx management in assisted reproduction clinics. A cross-sectional, internet-based survey of 442 members of Society for Reproductive Endocrinology and Infertility or Society of Reproductive Surgeons was performed. Physicians practising in states without infertility insurance mandates were more likely to report performing diagnostic surgery after an inconclusive hysterosalpingogram than physicians practising in states with mandates (RR 1.2, 95% CI 1.1-1.3, P < 0.01). Additionally, respondents in states without mandates were more likely to report that, due to lack of infertility insurance coverage, they did not perform salpingectomy (SPX) or proximal tubal occlusion (PTO) before assisted reproduction treatment (RR 1.4, 95% CI 1.1-1.8, P = 0.01). Finally, respondents in states without mandates were less likely to report that the presence of assisted reproduction treatment coverage determined the urgency with which they pursued SPX or PTO before treatment (RR 0.7, 95% CI 0.5-1.0, NS). These results persisted after controlling for physician years in practice, age and clinic volume. In conclusion, self-reported physician practice interventions for hydrosalpinges before assisted reproduction treatment may be associated with state-mandated infertility insurance. Fallopian tube dysfunction is a known cause of infertility and severe dysfunction is manifested by dilation and occlusion, known as hydrosalpinx. Outcomes with assisted reproductive techniques (ART) are lower when hydrosalpinges are present and while there are several theories for this, reproductive specialist recommend "neutralizing" the tube either by occlusion or removal in order to enhance pregnancy rates. In the United States, coverage for infertility services is not uniform with only 15 states having some legislation requiring infertility benefits. Some states where ART is covered liberally, physicians might have different practice patterns related to the neutralization of hydrosalpinges compared to those who are in non -mandated states. We utilized a survey of over 400 providers in the United States to examine their practice patterns as it relates to hydrosalpinges based on which state they practice in and whether or not that state has mandated coverage of not.

Axillary schwannoma with extensive cystic degeneration.

Schwannoma affect mainly head, neck, and flexor aspect of the limbs. Neurogenic tumors arising from the brachial plexus are rare and axillary schwannoma is extremely uncommon. Cystic degeneration is common in longstanding cases and which when aspirated may yield only macrophages or lymphocytes leading to false diagnosis of the case in spite of strong clinical suspicion. We report one such rare case of a solitary axillary schwannoma with extensive cystic degeneration, which was misdiagnosed on fine needle aspiration cytology and subsequently confirmed by the histopathological examination and immunohistochemistry.

Where do I go? A trauma victim's plea in an informal trauma system.

BACKGROUND: The three pillars of a good trauma system are the prehospital care, definitive care, and rehabilitative services. The prehospital care is a critical component of the efforts to lower trauma mortality. OBJECTIVE: To study the prehospital profile of patients who died due to trauma, compute the time taken to reach our facility, find the cause of delay, and make feasible recommendations. MATERIALS AND METHODS: A hospital-based study was performed at a trauma center in Puducherry from June 2009 to August 2010. Puducherry is a union territory of India in the geographical terrain of the state of Tamil Nadu. A total of 241deaths due to trauma were included. Apart from the demographic and injury characteristics, a detailed prehospital log was constructed regarding the time of incident, the referral patterns, care given in the prehospital phase, the distance travelled, and the total time taken to reach our center. RESULTS: The majority (59%) of patients were referred, with stopovers at two consecutive referral centers (30%), needing at least two vehicles to transport to definitive care (70%), clocking unnecessary distances (67%), and delayed due to non therapeutic intervention (87%). The majority of deaths (66%) were due to head injury. Only 2.96% of referred cases reached us within the first hour. Few of the patients coming directly to us had vehicle change due to local availability and lack of knowledge of predestined definitive care facility. Overall, 94.6% of direct cases arrived within 4 h whereas 93.3% of referred cases required up to 7 h to arrive at definitive care. CONCLUSIONS: Seriously injured patients lose valuable prehospital time because there is no direction regarding destination and interfacility transfer, a lack of seamless transport, and no concept of initial trauma care. The lack of direction is compounded in geographical areas that are situated at the border of political jurisdictions.

Endometrin as luteal phase support in assisted reproduction.

OBJECTIVE: To compare clinical pregnancy rate (PR) and live birth rate (LBR) between Endometrin monotherapy versus Endometrin and P in oil combination therapy in assisted reproductive technology (ART) cycles. DESIGN: Retrospective analysis. SETTING: Large private practice. PATIENT(S): Patients undergoing autologous fresh IVF cycles, autologous frozen ET cycles, and fresh oocyte donor cycles were included for analysis. INTERVENTION(S): Endometrin as a single agent for luteal support, Endometrin monotherapy or Endometrin with P in oil used at least once every 3 days for luteal support, Endometrin combination therapy. MAIN OUTCOME MEASURE(S): Clinical PR and LBR. RESULT(S): A total of 1,034 ART cycles were analyzed. Endometrin monotherapy was used in 694 of 1,034 (67%) cycles and Endometrin combination therapy was used in 340 of 1,034 (33%) cycles. In all fresh cycles, clinical PR was not significantly different (IVF autologous: Endometrin monotherapy 46.9% vs. Endometrin combination therapy 55.6%; donor oocyte endometrin monotherapy 45.2% vs. Endometrin combination therapy 52.0%). Frozen ET cycles had a significantly higher clinical PR and LBR with combination therapy group compared with monotherapy (clinical PR 47.9% vs. 23.5%; LBR 37.5% vs. 17.3%). CONCLUSION(S): Endometrin monotherapy was sufficient for the P component of luteal support and provided high PRs for fresh cycles in both autologous and donor oocyte cycles. Clinical PR and LBR in frozen ET cycles were significantly improved with the addition of IM P to Endometrin therapy. This may reflect the fact that lesser quality embryos are transferred in frozen ET cycles, and more intense P support is required for comparable PRs.

How members of the Society for Reproductive Endocrinology and Infertility and Society of Reproductive Surgeons evaluate, define, and manage hydrosalpinges.

OBJECTIVE: To describe the management of hydrosalpinges among Society for Reproduction Endocrinology and Infertility (SREI)/Society of Reproductive Surgeons (SRS) members. DESIGN: Cross-sectional survey of SREI/SRS members. SETTING: Academic and private practice-based reproductive medicine physicians. PARTICIPANT(S): A total of 442 SREI and/or SRS members. INTERVENTION(S): Internet-based survey. MAIN OUTCOME MEASURE(S): To understand how respondents evaluate, define, and manage hydrosalpinges. RESULT(S): Of 1,070 SREI and SRS members surveyed, 442 responded to all items, for a 41% response rate. Respondents represented both academic and private practice settings, and differences existed in the evaluation and management of hydrosalpinges. More than one-half (57%) perform their own hysterosalpingograms (HSGs), and 54.5% involve radiologists in their interpretation of tubal disease. Most respondents thought that a clinically significant hydrosalpinx on HSG is one that is distally occluded (80.4%) or visible on ultrasound (60%). Approximately one in four respondents remove a unilateral hydrosalpinx before controlled ovarian hyperstimulation (COH)/intrauterine insemination (IUI) and clomiphene citrate (CC)/IUI (29.3% and 22.8%, respectively), and physicians in private practice were more likely to intervene (COH: risk ratio [RR] 1.81, 95% confidence interval [CI] 1.31-2.51; CC: RR 1.98, 95% CI 1.33-2.95). Although laparoscopic salpingectomy was the preferred method of surgical management, nearly one-half responded that hysteroscopic tubal occlusion should have a role as a primary method of intervention. CONCLUSION(S): SREI/SRS members define a "clinically significant hydrosalpinx" consistently, and actual practice among members reflects American Society for Reproductive Medicine/SRS recommendations, with variation attributed to individual patient needs. Additionally, one in four members intervene before other infertility treatments when there is a unilateral hydrosalpinx present.

Video-assisted thoracoscopy in trauma: Case report and review of literature.

Video Assisted Thoracoscopy (VATS) like any other minimal access surgery has the obvious advantage of less surgical insult, enabling quicker recovery with its attendant reduced hospital costs and earlier return to work. The benefit of earlier return to work is much more important in developing countries. Unfortunately minimal access surgery is yet to gain popularity in trauma surgery, especially in the developing countries due to various reasons. We describe one case report where VATS was used successfully to remove an intrapleural foreign body.

Temporal distributions of anthropogenic Al, Zn and Pb in Hong Kong Porites coral during the last two centuries.

A 182-year long record of trace metal concentrations of aluminum, zinc and lead was reconstructed from a massive Porites coral skeleton from southeastern Hong Kong to evaluate the impacts of anthropogenic activity on the marine environment. Zn/Ca and Pb/Ca ratios fluctuate synchronously from the early 19th century to the present, indicating that the marine environment has been anthropogenically influenced since industrialization. Additionally, land reclamation, mining, and ship building activities are recorded by elevated Al/Ca ratios from 1900 to 1950. The coral record indicates that high levels of Zn, Pb and Al occur coincidentally with local wars, and may have contributed to partial colony mortality. Pb/Ca does not correlate well with hemispheric proxy records after 1950, indicating that coastal corals may be recording local rather than hemispheric contamination. Pb/Ca levels in Hong Kong, Guangdong and Hainan corals imply a continuous supply of Pb-based contamination to southern China not reflected in hemispheric signals.

Embryo transfer practices in the United States: a survey of clinics registered with the Society for Assisted Reproductive Technology.

OBJECTIVE: To gain a better understanding of factors influencing clinicians' embryo transfer practices. DESIGN: Cross-sectional survey. SETTING: Web-based survey conducted in December 2008 of individuals practicing IVF in centers registered with the Society for Assisted Reproductive Technology (SART). PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Prevalence of clinicians reporting following embryo transfer guidelines recommended by the American Society for Reproductive Medicine (ASRM), prevalence among these clinicians to deviate from ASRM guidelines in commonly encountered clinical scenarios, and practice patterns related to single embryo transfer. RESULT(S): Six percent of respondents reported following their own, independent guidelines for the number of embryos to transfer after IVF. Of the 94% of respondents who reported routinely following ASRM embryo transfer guidelines, 52% would deviate from these guidelines for patient request, 51% for cycles involving the transfer of frozen embryos, and 70% for patients with previously failed IVF cycles. All respondents reported routinely discussing the risks of multiple gestations associated with standard embryo transfer practices, whereas only 34% reported routinely discussing single embryo transfer with all patients. CONCLUSION(S): Although the majority of clinicians responding to our survey reported following ASRM embryo transfer guidelines, at least half would deviate from these guidelines in a number of different situations.

Lower chance of pregnancy with repeated cycles with in vitro fertilization.

OBJECTIVE: To evaluate pregnancy outcomes in initial and replicate IVF cycles. STUDY DESIGN: A retrospective analysis of 2,167 fresh, nondonor IVF cycles performed in a large private practice from January 1, 2005, to March 1, 2006. Standard controlled ovarian hyperstimulation and laboratory protocols were followed. RESULTS: Patients undergoing multiple treatment cycles were significantly older. There was no difference in body mass index or percentage of cancelled cycles with increasing number of IVF attempts. The number of retrieved, mature and fertilized oocytes progressively declined as the number of treatment cycles increased. The number of embryos transferred increased with increasing number of treatment cycles. Implantation, pregnancy and clinical pregnancy rates decreased significantly with the second treatment cycle and more markedly with 3-5 treatment cycles. CONCLUSION: The likelihood of a successful outcome declined with each additional treatment cycle. The most notable decrease in clinical pregnancy rates occurred after the third cycle. Patients who fail to conceive after 3 cycles of IVF should be counseled to begin considering other options.

Body mass index: impact on IVF success appears age-related.

BACKGROUND: The objective of this study was to examine the effect of BMI on IVF outcomes. METHODS: This was a retrospective analysis of all patients undergoing IVF from 1st January 2005 to 1st March 2006 in a large private practice using a single IVF laboratory. The patients underwent standard protocols for controlled ovarian hyperstimulation and embryology parameters. The main outcome measure was clinical pregnancy rate. RESULTS: A total of 2167 fresh, non-donor IVF cycles were queried, but to minimize bias, only the first treatment cycle for each patient was analyzed (n = 1273). The data were examined by multiple regression models that included BMI and Age as main effects plus a BMI x Age interaction. When examined as a main effect, BMI did not appear to have a major effect on IVF outcome, but there was a significant BMI x Age interaction. At younger ages, a high BMI had a pronounced negative influence on fertility, but this effect diminished as the patient age increased. Clinical pregnancy rates decreased with increasing BMI and increasing Age. CONCLUSIONS: In younger patients undergoing IVF, BMI has a significant negative impact on fertility that diminishes as patients reach their mid thirties. After Age 36, BMI has a minimal impact on fertility.

Adding human menopausal gonadotrophin to antagonist protocols - is there a benefit?

The objective of this retrospective analysis was to compare the clinical outcomes of recombinant FSH (r-FSH) with combination r-FSH plus human menopausal gonadotrophin (HMG) protocols in a large private practice using a single IVF laboratory, from 2001 to 2003. Patients underwent ovarian stimulation by standard gonadotrophin-releasing hormone (GnRH) antagonist protocol using r-FSH or combination r-FSH plus HMG. When two or more follicles had attained a minimum mean diameter of 20 mm, follicular triggering was achieved with either recombinant HCG (r-HCG; Ovidrel, 250 microg s.c.) or urinary HCG (u-HCG; 10,000 IU i.m.). The main outcome measures were number of oocytes retrieved and clinical pregnancy rate. There was a lower percentage of cancelled cycles and an increased number of oocytes retrieved, mature oocytes, oocytes that fertilized, embryo that cleaved and a tendency towards higher clinical pregnancy rates in patients treated with r-FSH alone compared with those treated with r-FSH plus HMG. Patients treated with r-FSH plus HMG had lower miscarriage rates and the live birth rate was similar in both treatment groups. In conclusion, irrespective of age, using a treatment regimen consisting of a combination of HMG plus r-FSH was not beneficial compared with r-FSH alone in patients using a GnRH antagonist protocol.

Age-matched comparison of recombinant and urinary HCG for final follicular maturation.

This age-matched retrospective analysis compared the clinical outcomes of recombinant human chorionic gonadotrophin (rHCG) and urinary HCG (uHCG) in patients undergoing fresh, nondonor IVF cycles. The patients underwent ovarian stimulation by standard gonadotrophin-releasing hormone (GnRH) agonist down-regulation or a GnRH antagonist protocol using recombinant FSH (rFSH) alone or in combination with human menopausal gonadotrophin. When two or more follicles had attained a mean diameter of 20 mm, follicular triggering was achieved with either Ovidrel (rHCG) 250 mug SC or uHCG 10,000 IU IM. Patients receiving rHCG were considered subjects, and they were age-matched in a 1:2 ratio to patients receiving uHCG, who were designated as controls. The main outcome measures were number of oocytes retrieved, number of mature oocytes obtained, number of oocytes fertilized and clinical pregnancy rates. A total of 273 subjects were age-matched and compared with 546 controls. Recombinant HCG had a minimal effect on the number of oocytes retrieved (13.4 versus 13.2), mature oocytes (10.5 versus 10.3) and oocytes fertilized (8.2 versus 7.8) compared with uHCG. Pregnancy (46.0 versus 45.2%) and clinical pregnancy rates (38.1 versus 36.8%) were similar for rHCG and uHCG. Recombinant HCG was as effective as uHCG for final follicular maturation in IVF cycles.