PlGF blockade does not inhibit angiogenesis during primary tumor growth.

It has been recently reported that treatment with an anti-placenta growth factor (PlGF) antibody inhibits metastasis and primary tumor growth. Here we show that, although anti-PlGF treatment inhibited wound healing, extravasation of B16F10 cells, and growth of a tumor engineered to overexpress the PlGF receptor (VEGFR-1), neutralization of PlGF using four novel blocking antibodies had no significant effect on tumor angiogenesis in 15 models. Also, genetic ablation of the tyrosine kinase domain of VEGFR-1 in the host did not result in growth inhibition of the anti-VEGF-A sensitive or resistant tumors tested. Furthermore, combination of anti-PlGF with anti-VEGF-A antibodies did not result in greater antitumor efficacy than anti-VEGF-A monotherapy. In conclusion, our data argue against an important role of PlGF during primary tumor growth in most models and suggest that clinical evaluation of anti-PlGF antibodies may be challenging.

Targeting PTPRK-RSPO3 colon tumours promotes differentiation and loss of stem-cell function.

Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

Accumulation of integrase strand transfer inhibitor resistance mutations confers high-level resistance to dolutegravir in non-B subtype HIV-1 strains from patients failing raltegravir in Uganda.

BACKGROUND: Increasing first-line treatment failures in low- and middle-income countries (LMICs) have led to increased use of integrase strand transfer inhibitors (INSTIs) such as dolutegravir. However, HIV-1 susceptibility to INSTIs in LMICs, especially with previous raltegravir exposure, is poorly understood due to infrequent reporting of INSTI failures and testing for INSTI drug resistance mutations (DRMs). METHODS: A total of 51 non-subtype B HIV-1 infected patients failing third-line (raltegravir-based) therapy in Uganda were initially selected for the study. DRMs were detected using Sanger and deep sequencing. HIV integrase genes of 13 patients were cloned and replication capacities (RCs) and phenotypic susceptibilities to dolutegravir, raltegravir and elvitegravir were determined with TZM-bl cells. Spearman's correlation coefficient was used to determine cross-resistance between INSTIs. RESULTS: INSTI DRMs were detected in 47% of patients. HIV integrase-recombinant virus carrying one primary INSTI DRM (N155H or Y143R/S) was susceptible to dolutegravir but highly resistant to raltegravir and elvitegravir (>50-fold change). Two patients, one with E138A/G140A/Q148R/G163R and one with E138K/G140A/S147G/Q148K, displayed the highest reported resistance to raltegravir, elvitegravir and even dolutegravir. The former multi-DRM virus had WT RC whereas the latter had lower RCs than WT. CONCLUSIONS: In HIV-1 subtype A- and D-infected patients failing raltegravir and harbouring INSTI DRMs, there is high-level resistance to elvitegravir and raltegravir. More routine monitoring of INSTI treatment may be advised in LMICs, considering that multiple INSTI DRMs may have accumulated during prolonged exposure to raltegravir during virological failure, leading to high-level INSTI resistance, including dolutegravir resistance.

Seroprevalence of Q Fever in Patients Undergoing Heart Valve Replacement Surgery.

BACKGROUND AND AIM OF THE STUDY: Q fever, caused by the rickettsia Coxiella burnetii, is a worldwide zoonotic disease with both acute and chronic manifestations. Endocarditis is the principal chronic manifestation. Q fever can easily be mistaken for degenerative valve disease due to its indolent presentation, the fastidious nature of the organism (routine cultures are negative), and the absence of a typical echocardiographic and macroscopic appearance for endocarditis. Prosthetic valve failure, with associated morbidity and mortality, have been described following unrecognized infections. METHODS: Previous studies have documented the value of screening strategies in areas of high prevalence. Hence, a pilot study was conducted in a low-prevalence setting, in which 139 patients at two tertiary cardiac centers attending for elective valve replacement for degenerative valvular disease underwent testing for chronic Q fever infection by serological and molecular methods on blood and valve tissue. RESULTS: Five patients (3.7%) had serological evidence of past exposure to Q fever (consistent with rates in the literature). None had evidence of chronic Q fever endocarditis. The cost of adopting a universal screening strategy is around £40,000 per case (if serology is used to screen patients prior to surgery). CONCLUSIONS: Alternative and more cost-effective methods for identifying clinically quiet cases of chronic Q fever endocarditis are required.

Building intentions with the theory of planned behaviour: a qualitative assessment of salient beliefs about pharmacy value added services in Malaysia.

OBJECTIVE: To improve pharmaceutical care delivery in Malaysia, the Ministry of Health (MOH) had introduced the concept of value added services (VAS). Despite its reported convenience and advantages, VAS utilization rate is low in the country. The study aims to explore patients' understanding, beliefs and expectations towards VAS in Malaysia using the theory of planned behaviour (TPB) as the theoretical model. METHODS: A qualitative methodology was used whereby face-to-face interviews were conducted with 12 patients who collected partial medicine supplies from government pharmacies. Participants were recruited using purposive and snowball sampling method in the state of Negeri Sembilan, Malaysia. Interviews were audio-recorded. Verbatim transcription and thematic content analysis were performed on the data. RESULTS: Thematic content analysis yielded five major themes: (i) attitudes towards using VAS, (ii) subjective norms, (iii) perceived behavioural control, (iv) lack of knowledge and understanding of VAS and (v) expectations towards VAS. CONCLUSION: The interviews explored and informed new information about salient beliefs towards pharmacy VAS. The findings suggest that VAS is still in its infancy and a more robust and effective advertising and marketing campaign is needed to boost the adoption rate. Behavioural attitudes, subjective norms and perceived control elements were discussed and serve as important variables of interest in future study. Expectations towards VAS serve as an important guideline to further improve patient-oriented services.

Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel.

Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing.

Perioperative transesophageal echocardiography for aortic dissection.

PURPOSE: Aortic dissection is an infrequent but serious condition that often requires immediate operative intervention. We explore recent developments in the classification of aortic dissection and perioperative transesophageal echocardiography that assist with quantifying the severity of disease and facilitate its management. PRINCIPAL FINDINGS: We describe the pivotal role of echocardiography in relation to key surgical considerations such as cannulation, aortic root surgery, perfusion in the aortic arch vessels, stenting in hybrid arch repair, and timing of preventative surgery. CONCLUSION: Developments in the classification of aortic dissection have improved our perspective and understanding of the key presenting features that affect mortality. Improvements in patient outcome may be achieved in part by appropriately timed echocardiography-guided surgery.

Hearing dummies: individualized computer models of hearing impairment.

UNLABELLED: Objective: Our aim was to explore the usage of individualized computer models to simulate hearing loss based on detailed psychophysical assessment and to offer hypothetical diagnoses of the underlying pathology. DESIGN: Individualized computer models of normal and impaired hearing were constructed and evaluated using the psychophysical data obtained from human listeners. Computer models of impaired hearing were generated to reflect the hypothesized underlying pathology (e.g. dead regions, outer hair cell dysfunction, or reductions in endocochlear potential). These models were evaluated in terms of their ability to replicate the original patient data. STUDY SAMPLE: Auditory profiles were measured for two normal and five hearing-impaired listeners using a battery of three psychophysical tests (absolute thresholds, frequency selectivity, and compression). RESULTS: The individualized computer models were found to match the data. Useful fits to the impaired profiles could be obtained by changing only a single parameter in the model of normal hearing. Sometimes, however, it was necessary to include an additional dead region. CONCLUSION: The creation of individualized computer models of hearing loss can be used to simulate auditory profiles of impaired listeners and suggest hypotheses concerning the underlying peripheral pathology.

The risk of developing pseudomyxoma peritonei from a non-perforated low grade appendiceal mucinous neoplasm found at appendicectomy.

INTRODUCTION: Low-grade appendiceal mucinous neoplasms (LAMNs) are classified as non-perforated (pTis, pT3) or perforated (pT4), and considered precursors of pseudomyxoma peritonei (PMP). This study aims to quantify the risk of developing PMP from pTis and pT3 LAMNs. MATERIALS AND METHODS: Retrospective analysis of a prospectively collected database identified LAMN patients referred to a specialist centre from 2004 to 2019. pT4 LAMNs and other appendix tumours were excluded. All patients had specialist review of their pathology, operation note, and a CT scan (at least 6 weeks post-operatively). Surveillance CTs were then performed at 6, 12, 24, 36, 48, & 60 months, with tumour markers (CEA, CA19-9, CA125). RESULTS: 193 pT3/pTis LAMN patients were included (pTis = 153, pT3 = 40). Median follow-up = 6.45 (3.91-22.13) years, M:F ratio = 1:1.57, and median age = 57 (23-83) years. Initial surgery included: appendicectomy (67 %), appendicectomy + visceral resection (6 %), and right hemicolectomy (27 %). R1 resections were identified in 5/193 patients (2.5 %). 3 R1 patients underwent re-operation (2 caecal pole excision and 1 ileocecectomy), none of which had residual tumour. 8/193 patients (4 %) were lost to follow up. None of the remaining 185 developed PMP. CONCLUSION: This is the largest reported series of pTis/pT3 LAMNs with standardised follow-up in the literature. LAMNs correctly classified as pT3/pTis (after careful specialist review of pathology, operation note, and a baseline post-operative CT) have negligible risk of developing PMP and should have low intensity surveillance. If completely excised, further surgery is not indicated. R1 resections should be considered on an individual basis at a specialist centre.

The melanosomal protein PMEL17 as a target for antibody drug conjugate therapy in melanoma.

Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing in vitro and in vivo.

A computer model of the auditory periphery and its application to the study of hearing.

Computer models of the auditory periphery provide a tool for -formulating theories concerning the relationship between the physiology of the auditory system and the perception of sounds both in normal and impaired hearing. However, the time-consuming nature of their construction constitutes a major impediment to their use, and it is important that transparent models be available on an 'off-the-shelf' basis to researchers. The MATLAB Auditory Periphery (MAP) model aims to meet these requirements and be freely available. The model can be used to simulate simple psychophysical tasks such as absolute threshold, pitch matching and forward masking and those used to measure compression and frequency selectivity. It can be used as a front end to automatic speech recognisers for the study of speech in quiet and in noise. The model can also simulate theories of hearing impairment and be used to make predictions about the efficacy of hearing aids. The use of the software will be described along with illustrations of its application in the study of the psychology of hearing.

Phosphatidylserine receptor Tim-4 is essential for the maintenance of the homeostatic state of resident peritoneal macrophages.

Tim-4 is a phosphatidylserine (PS) receptor that is expressed on various macrophage subsets. It mediates phagocytosis of apoptotic cells by peritoneal macrophages. The in vivo functions of Tim-4 in phagocytosis and immune responses, however, are still unclear. In this study, we show that Tim-4 quickly forms punctate caps on contact with apoptotic cells, in contrast to its normal diffused expression on the surface of phagocytes. Despite its expression in marginal zone and tingible body macrophages, Tim-4 deficiency only minimally affects outcomes of several acute immune challenges, including the trapping of apoptotic cells in the marginal zone, the clearance apoptotic cells by tingible body macrophages, and the formation of germinal centers and elicitation of antibody responses against sheep red blood cells (SRBCs). In addition, Tim-4(-/-) resident peritoneal macrophages (rPMs) phagocytose necrotic cells and other opsonized targets normally. However, their ability to bind and engulf apoptotic cells is significantly compromised both in vitro and in vivo. Most importantly, Tim-4 deficiency results in increased cellularity in the peritoneum. Resting rPMs produce higher TNF-alpha in culture. Their response to LPS, on the contrary, is dampened. Our data support an indispensible role of Tim-4 in maintaining the homeostasis of rPMs.

Acquisition of auditory profiles for good and impaired hearing.

OBJECTIVE: The aim of this study was to develop a user-friendly way of measuring patients' threshold and supra-threshold hearing, with potential for application in clinical research. The end-product of these tests is a graphical profile summarizing absolute threshold, frequency selectivity, and compression characteristics across a spectrum of frequencies (0.25-6 kHz). DESIGN: A battery of three psychophysical hearing tests consisted of measures of absolute threshold, frequency selectivity, and compression. An automated, cued, single-interval, adaptive tracking procedure was employed. The tests results were collated and used to generate a readily visualized 'profile' for each listener. STUDY SAMPLE: Participants were 83 adults (57 impaired-hearing and 26 good-hearing, age 20-75 years). RESULTS: Listeners tolerated the tests well. Single-ear profiles were obtained in an average of 74 minutes testing time (range 46-120 minutes). The variability of individual measurements was low. Substantial differences between normal and impaired listeners and also among the impaired listeners were observed. Qualitative differences in compression and frequency-selectivity were seen that could not be predicted by threshold measurements alone. CONCLUSIONS: The hearing profiles are informative with respect to supra-threshold hearing performance and the information is easily accessible through the graphical display. Further development is required for routine use in a clinical context.

Endophthalmitis: Microbiology and Organism Identification Using Current and Emerging Techniques.

Endophthalmitis is an ophthalmological emergency requiring timely and appropriate diagnosis and treatment. Microbiological methods of microscopy (Gram's staining) and culture are the current gold standard for organism identification. However, a significant proportion of endophthalmitis remains culture-negative-perhaps the inflammation is non-infectious in origin, results from a novel organism are unidentifiable or because the causative organism is non-culturable often due to pre-treatment with antibiotics. This review outlines the microbiological profile of endophthalmitis, current clinically used methods for organism identification, and the newer molecular techniques of polymerase chain reaction (PCR) and next-generation sequencing (NGS) technology as diagnostic tools for endophthalmitis. They offer the potential to improve organism identification rates and clinical outcomes in infectious diseases, representing an exciting future direction for organism identification in endophthalmitis. Based on the largest ophthalmic hospital in Australia, we highlight the key practical challenges faced by Australian diagnostic laboratories for their use in a clinical setting.

Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis.

Haploinsufficiency of Dll4, a vascular-specific Notch ligand, has shown that it is essential for embryonic vascular development and arteriogenesis. Mechanistically, it is unclear how the Dll4-mediated Notch pathway contributes to complex vascular processes that demand meticulous coordination of multiple signalling pathways. Here we show that Dll4-mediated Notch signalling has a unique role in regulating endothelial cell proliferation and differentiation. Neutralizing Dll4 with a Dll4-selective antibody rendered endothelial cells hyperproliferative, and caused defective cell fate specification or differentiation both in vitro and in vivo. In addition, blocking Dll4 inhibited tumour growth in several tumour models. Remarkably, antibodies against Dll4 and antibodies against vascular endothelial growth factor (VEGF) had paradoxically distinct effects on tumour vasculature. Our data also indicate that Dll4-mediated Notch signalling is crucial during active vascularization, but less important for normal vessel maintenance. Furthermore, unlike blocking Notch signalling globally, neutralizing Dll4 had no discernable impact on intestinal goblet cell differentiation, supporting the idea that Dll4-mediated Notch signalling is largely restricted to the vascular compartment. Therefore, targeting Dll4 might represent a broadly efficacious and well-tolerated approach for the treatment of solid tumours.

Phosphine-catalyzed regio- and stereo-selective hydroboration of ynamides to (Z)-ß-borylenamides.

We report a tri-n-butyl phosphine catalyzed regio- and stereo-selective hydroboration of ynamides to yield (Z)-ß-borylenamides in good yields. Surprisingly, a formal cis addition to the triple bond was observed as confirmed by NMR and X-ray crystallography. 31P NMR studies suggest that a zwitterionic vinylphosphonium intermediate is key in the mechanism. The resulting products were further transformed to ß-CF3 enamides via stereoretentive trifluoromethylation.

Surgical Techniques for Tonsillectomy and Perioperative Respiratory Complications in Children.

OBJECTIVES: The aim of this study was to determine the incidence of perioperative respiratory complications in children following tonsillectomy with cold and hot dissection surgical techniques. STUDY DESIGN: The study was a retrospective cohort study. SETTING: Retrospective chart review was performed for all children presenting for a tonsillectomy at Texas Children's Hospital from November 2015 to December 2017. METHODS: Pre- and intraoperative patient factors, including surgical technique with cold or hot dissection (electrocautery or radiofrequency ablation), and perioperative anesthetic factors were collected to determine the incidence of perioperative respiratory complications. RESULTS: A total of 2437 patients underwent a tonsillectomy at Texas Children's Hospital from November 2015 to December 2017. The incidence of perioperative respiratory complications was 20.0% (n = 487). Sickle cell disease, cardiac disease, reactive airway disease, pulmonary disease, age >2 and <3 years, and obesity, defined as a body mass index >95th percentile for age, were significant for overall perioperative respiratory complications. There was no difference in the incidence of perioperative respiratory complications in children undergoing tonsillectomy by cold or hot dissection. CONCLUSION: Perioperative respiratory complications following tonsillectomy are more affected by patient factors than surgical technique.

Nanoscale resolution, multicomponent biomolecular arrays generated by aligned printing with parylene peel-off.

We present "Print-and-Peel", a high-throughput method to generate multicomponent biomolecular arrays with sub-100 nm nanoscale feature width. An inkjet printer is first aligned to a parylene template containing nanoscale openings. After printing, the parylene is peeled off to reveal uniformly patterned nanoscale features, despite the imperfect morphologies of the original inkjet spots. We further patterned combinatorial nanoarrays by performing a second print-run superimposed over the first, thereby extending the multiplexing capability of the technique.