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Androgen Receptor (AR) alterations (amplification, point mutations, and splice variants) are master players in metastatic castration resistant prostate cancer (CRPC) progression and central therapeutic targets for patient management. Here, we have developed two multiplexed droplet digital PCR (ddPCR) assays to detect AR copy number (CN) and the key point mutation T877A. Overcoming challenges of determining gene amplification from liquid biopsies, these assays cross-validate each other to produce reliable AR amplification and mutation data from plasma cell free DNA (cfDNA) of advanced prostate cancer (PC) patients. Analyzing a mixed PC patient cohort consisting of CRPC and hormone sensitive prostate cancer (HSPC) patients showed that 19% (9/47) patients had AR CN amplification. As expected, only CRPC patients were positive for AR amplification, while interestingly the T877A mutation was identified in two patients still considered HSPC at the time. The ddPCR based analysis of AR alterations in cfDNA is highly economic, feasible, and informative to provide biomarker detection that may help to decide on the best follow-up therapy for CRPC patients.

Respirable aerosols (< 5 µm in diameter) present a high risk of SARS-CoV-2 transmission. Guidelines recommend using aerosol precautions during aerosol-generating procedures, and droplet (> 5 µm) precautions at other times. However, emerging evidence indicates respiratory activities may be a more important source of aerosols than clinical procedures such as tracheal intubation. We aimed to measure the size, total number and volume of all human aerosols exhaled during respiratory activities and therapies. We used a novel chamber with an optical particle counter sampling at 100 l.min -1 to count and size-fractionate close to all exhaled particles (0.5-25 µm). We compared emissions from ten healthy subjects during six respiratory activities (quiet breathing; talking; shouting; forced expiratory manoeuvres; exercise; and coughing) with three respiratory therapies (high-flow nasal oxygen and single or dual circuit non-invasive positive pressure ventilation). Activities were repeated while wearing facemasks. When compared with quiet breathing, exertional respiratory activities increased particle counts 34.6-fold during talking and 370.8-fold during coughing (p < 0.001). High-flow nasal oxygen 60 at l.min -1 increased particle counts 2.3-fold (p = 0.031) during quiet breathing. Single and dual circuit non-invasive respiratory therapy at 25/10 cm.H 2 O with quiet breathing increased counts by 2.6-fold and 7.8-fold, respectively (both p < 0.001). During exertional activities, respiratory therapies and facemasks reduced emissions compared with activities alone. Respiratory activities (including exertional breathing and coughing) which mimic respiratory patterns during illness generate substantially more aerosols than non-invasive respiratory therapies, which conversely can reduce total emissions. We argue the risk of aerosol exposure is underappreciated and warrants widespread, targeted interventions., (© 2021 Association of Anaesthetists.)

Healthcare workers are at risk of infection during the severe acute respiratory syndrome coronavirus-2 pandemic. International guidance suggests direct droplet transmission is likely and airborne transmission occurs only with aerosol-generating procedures. Recommendations determining infection control measures to ensure healthcare worker safety follow these presumptions. Three mechanisms have been described for the production of smaller sized respiratory particles ('aerosols') that, if inhaled, can deposit in the distal airways. These include: laryngeal activity such as talking and coughing; high velocity gas flow; and cyclical opening and closure of terminal airways. Sneezing and coughing are effective aerosol generators, but all forms of expiration produce particles across a range of sizes. The 5-μm diameter threshold used to differentiate droplet from airborne is an over-simplification of multiple complex, poorly understood biological and physical variables. The evidence defining aerosol-generating procedures comes largely from low-quality case and cohort studies where the exact mode of transmission is unknown as aerosol production was never quantified. We propose that transmission is associated with time in proximity to severe acute respiratory syndrome coronavirus-1 patients with respiratory symptoms, rather than the procedures per se. There is no proven relation between any aerosol-generating procedure with airborne viral content with the exception of bronchoscopy and suctioning. The mechanism for severe acute respiratory syndrome coronavirus-2 transmission is unknown but the evidence suggestive of airborne spread is growing. We speculate that infected patients who cough, have high work of breathing, increased closing capacity and altered respiratory tract lining fluid will be significant producers of pathogenic aerosols. We suggest several aerosol-generating procedures may in fact result in less pathogen aerosolisation than a dyspnoeic and coughing patient. Healthcare workers should appraise the current evidence regarding transmission and apply this to the local infection prevalence. Measures to mitigate airborne transmission should be employed at times of risk. However, the mechanisms and risk factors for transmission are largely unconfirmed. Whilst awaiting robust evidence, a precautionary approach should be considered to assure healthcare worker safety., (© 2020 Association of Anaesthetists.)

Circulating tumour cells (CTCs) are an emerging resource for monitoring cancer biomarkers. New technologies for CTC isolation and biomarker detection are increasingly sensitive, however, the ideal blood storage conditions to preserve CTC-specific mRNA biomarkers remains undetermined. Here we tested the preservation of tumour cells and CTC-mRNA over time in common anticoagulant ethylene-diamine-tetra-acetic acid (EDTA) and acid citrate dextrose solution B (Citrate) blood tubes compared to preservative-containing blood tubes. Blood samples spiked with prostate cancer cells were processed after 0, 24, 30, and 48 h storage at room temperature. The tumour cell isolation efficiency and the mRNA levels of the prostate cancer biomarkers androgen receptor variant 7 (AR-V7) and total AR, as well as epithelial cell adhesion molecule (EpCAM) were measured. Spiked cells were recovered across all storage tube types and times. Surprisingly, tumour mRNA biomarkers were readily detectable after 48 h storage in EDTA and Citrate tubes, but not in preservative-containing tubes. Notably, AR-V7 expression was detected in prostate cancer patient blood samples after 48 h storage in EDTA tubes at room temperature. This important finding presents opportunities for measuring AR-V7 expression from clinical trial patient samples processed within 48 h-a much more feasible timeframe compared to previous recommendations.

Androgen receptor splice variant V7 (AR-V7) was recently identified as a valuable predictive biomarker in metastatic castrate-resistant prostate cancer. Here, we report a new, sensitive and accurate screen for AR-V7 mRNA expression directly from circulating tumor cells (CTCs): We combined EpCAM-based immunomagnetic CTC isolation using the IsoFlux microfluidic platform with droplet digital polymerase chain reaction (ddPCR) to analyze total AR and AR-V7 expression from prostate cancer patients CTCs. We demonstrate that AR-V7 is reliably detectable in enriched CTC samples with as little as five CTCs, even considering tumor heterogeneity, and confirm detection of AR-V7 in CTC samples from advanced prostate cancer (PCa) patients with AR-V7 detection limited to castrate resistant disease status in our sample set. Sensitive molecular analyses of circulating tumor cells (CTCs) or circulating tumor nucleic acids present exciting strategies to detect biomarkers, such as AR-V7 from non-invasive blood samples, so-called blood biopsies.

beta-N-Acetylhexosaminidase was purified from the extract of cabbage by sequential steps of ammonium sulfate fractionation, chromatofocusing, DEAE-Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 HR gel filtration. By these steps, the purity of the enzyme increased by 256 fold with a recovery of 8%. The purified enzyme was homogeneous as examined by native PAGE. It showed an optimal pH of 4, an optimal temperature of 60 degrees C and a Km of 0.94 mM for hydrolysis of pNp-beta-GlcNAc. The molecular mass of the enzyme determined from filtration through Sephacryl S-200 was 150 kDa. Three subunits with molecular mass of 64, 57 and 51 kDa were observed as determined by SDS-PAGE. NBS (0.025 mM), DEPC (3 mM) and WRK (30 mM) significantly inhibited the activity of the enzyme. The enzyme also showed activity toward pNp-beta-GalNAc, N,N'-diacetylchitobiose, N,N',N"-triacetylchitotriose and N,N',N",N"'-tetraacetyl chitotetraose but showed no activity toward pNp-alpha-GlcNAc, chitin and ethylene glycol chitin.