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Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and β-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in maternal plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved. Due to the presence of background maternal DNA, which interferes with the analysis of fetal DNA in maternal plasma, noninvasive prenatal diagnosis of maternally inherited mutations has not been possible. Here we describe a digital relative mutation dosage (RMD) approach that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in maternal plasma. When applied to the testing of women heterozygous for the CD41142 (-C1Tr) and hemoglobin E mutations on HBB, digital RMD allows the fetal genotype to be deduced. The diagnostic performance of digital RMD is dependent on interplay between the fractional fetal DNA concentration and number of DNA molecules in maternal plasma. To achieve fetal genotype diagnosis at lower volumes of maternal plasma, fetal DNA enrichment is desired. We thus developed a digital nucleic acid size selection (NASS) strategy that effectively enriches the fetal DNA without additional plasma sampling or experimental time. We show that digital NASS can work in concert with digital RMD to increase the proportion of cases with classifiable fetal genotypes and to bring noninvasive prenatal diagnosis of monogenic diseases closer to reality. [ABSTRACT FROM AUTHOR]

Introduction: Cambodia's health equity fund (HEF) is the country's most significant social security scheme, covering the poorest one-fifth of the national population. During the last two decades, the HEF system was scaled up from an initial two health districts to national coverage of public health facilities. This is the first national study to examine the impact of the HEF on the utilisation of public health facilities., Methods: We first investigated the level of national HEF population coverage and health service use made by HEF eligible members using an administrative HEF operational dataset. Second, through multilevel interrupted time series analysis of routine monthly utilisation statistics during 2006-2013, we evaluated the impact of the HEF on hospital and health centre utilisation., Results: The proportion of HEF beneficiaries using hospital services in a given year (4.6%) appeared to exceed rates in the general population (3.3%). The introduction of the HEF was associated with: a significant level change in the monthly number of consultations at HCs followed by a gradual slope increase in time trend and a significant level change in the monthly number of deliveries. Overall, this was equivalent to a 15.6% net increase in number of consultations and 5.3% in deliveries in the first year. At RHs: a significant level change in the number of RH inpatient cases, followed by a sustained slope increase; a significant slope increase in the number of outpatient consultations and in the overall number of newborn deliveries. Overall, this was equivalent to a 47.9% net increase in inpatient cases, 24.1% in outpatient cases and 31.4% in deliveries in the first year., Conclusion: The implementation of the HEF scheme was associated with increased utilisation of primary and secondary care services by the poor., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Knowledge of the costs of health services improves health facility management and aids in health financing for universal health coverage. Because of resource requirements that are often not present in low- and middle-income countries, costing exercises are rare and infrequent. Here we report findings from the initial phase of establishing a routine costing system for health services implemented in three provinces in Cambodia., Methods: Data was collected for the 2016 financial year from 20 health centres (including four with beds) and five hospitals (three district hospitals and two provincial hospitals). The costs to the providers for health centres were calculated using step-down allocations for selected costing units, including preventive and curative services, delivery, and patient contact, while for hospitals this was complemented with bed-day and inpatient day per department. Costs were compared by type of facility and between provinces., Results: All required information was not readily available at health facilities and had to be recovered from various sources. Costs per outpatient consultation at health centres varied between provinces (from US$2.33 to US$4.89), as well as within provinces. Generally, costs were inversely correlated with the quantity of service output. Costs per contact were higher at health centres with beds than health centres without beds (US$4.59, compared to US$3.00). Conversely, costs for delivery were lower in health centres with beds (US$128.7, compared to US$413.7), mainly because of low performing health centres without beds. Costs per inpatient-day varied from US$27.61 to US$55.87 and were most expensive at the lowest level hospital., Conclusions: Establishing a routine health service costing system appears feasible if recording and accounting procedures are improved. Information on service costs by health facility level can provide useful information to optimise the use of available financial and human resources.

Background: Clinical trials management can be studied using project management theory. The CYCLE pilot randomized controlled trial (RCT) was conducted to determine the feasibility of a future rehabilitation trial of early in-bed cycling in the intensive care unit (ICU). In-bed cycling is a novel intervention, not typically available in ICUs. Implementation of this intervention requires personnel with specialized clinical expertise caring for critically ill patients and use of the in-bed cycle. Our objective was to describe the implementation and conduct of our pilot RCT using a project management approach., Methods: We retrospectively reviewed activities, timelines, and personnel involved in the trial. We organized activities into four project management phases: initiation, planning, execution, and monitoring and controlling. Data sources included Methods Centre documents used for trial coordination and conduct, and the trial data set. We report descriptive statistics as counts and proportions and also medians and quartiles, and we summarize the lessons learned., Results: Seven ICUs in Canada participated in the trial. Time from research ethics board and contracts submission to first enrolment was a median (first quartile, third quartile) of 185 (146, 209) and 162 (114, 181) days, respectively. We trained 128 personnel on the CYCLE pilot RCT protocol, and 80 (63%) completed trial-related activities. Four sites required additional training after start-up due to staff turnover and leaves of absence. Over 15 months, we screened 864 patients: 256 were eligible and 66 were enrolled. Despite an 85% consent rate, 74% (190/256) of eligible patients were not randomized, largely (80% [152/190]) due to physiotherapist availability. Thirteen percent of recruitment weeks were lost due to physiotherapist staffing shortages. We highlight five key lessons learned: (1) prepare and anticipate site needs; (2) communicate regularly; (3) proactively analyse and act on process measure data; (4) develop contingency plans; (5) express appreciation to participating sites., Conclusions: Our analysis highlights the scope of relevant activities, rigorous training and monitoring, number and types of required personnel, and time required to conduct a multicentre ICU rehabilitation intervention trial. Our lessons learned can help others interested in implementing complex intervention trials, such as rehabilitation., Trial Registration: ClinicalTrials.gov, NCT02377830 . Registered prospectively on 4 March 2015.

The rodlet structure present on the Aspergillus fumigatus conidial surface hides conidia from immune recognition. In spite of the essential biological role of the rodlets, the molecular basis for their self-assembly and disaggregation is not known. Analysis of the soluble forms of conidia-extracted and recombinant RodA by NMR spectroscopy has indicated the importance of disulfide bonds and identified two dynamic regions as likely candidates for conformational change and intermolecular interactions during conversion of RodA into the amyloid rodlet structure. Point mutations introduced into the RODA sequence confirmed that (1) mutation of a single cysteine was sufficient to block rodlet formation on the conidial surface and (2) both presumed amyloidogenic regions were needed for proper rodlet assembly. Mutations in the two putative amyloidogenic regions retarded and disturbed, but did not completely inhibit, the formation of the rodlets in vitro and on the conidial surface. Even in a disturbed form, the presence of rodlets on the surface of the conidia was sufficient to immunosilence the conidium. However, in contrast to the parental conidia, long exposure of mutant conidia lacking disulfide bridges within RodA or expressing RodA carrying the double (I115S/I146G) mutation activated dendritic cells with the subsequent secretion of proinflammatory cytokines. The immune reactivity of the RodA mutant conidia was not due to a modification in the RodA structure, but to the exposure of different pathogen-associated molecular patterns on the surface as a result of the modification of the rodlet surface layer. The full degradation of the rodlet layer, which occurs during early germination, is due to a complex array of cell wall bound proteases. As reported earlier, this loss of the rodlet layer lead to a strong anti- fumigatus host immune response in mouse lungs., (© 2019 The Authors.)

Introduction: Acute rehabilitation in critically ill patients can improve post-intensive care unit (post-ICU) physical function. In-bed cycling early in a patient's ICU stay is a promising intervention. The objective of this study was to determine the feasibility of recruitment, intervention delivery and retention in a multi centre randomised clinical trial (RCT) of early in-bed cycling with mechanically ventilated (MV) patients., Methods: We conducted a pilot RCT conducted in seven Canadian medical-surgical ICUs. We enrolled adults who could ambulate independently before ICU admission, within the first 4 days of invasive MV and first 7 days of ICU admission. Following informed consent, patients underwent concealed randomisation to either 30 min/day of in-bed cycling and routine physiotherapy (Cycling) or routine physiotherapy alone (Routine) for 5 days/week, until ICU discharge. Our feasibility outcome targets included: accrual of 1-2 patients/month/site; >80% cycling protocol delivery; >80% outcomes measured and >80% blinded outcome measures at hospital discharge. We report ascertainment rates for our primary outcome for the main trial (Physical Function ICU Test-scored (PFIT-s) at hospital discharge)., Results: Between 3/2015 and 6/2016, we randomised 66 patients (36 Cycling, 30 Routine). Our consent rate was 84.6 % (66/78). Patient accrual was (mean (SD)) 1.1 (0.3) patients/month/site. Cycling occurred in 79.3% (146/184) of eligible sessions, with a median (IQR) session duration of 30.5 (30.0, 30.7) min. We recorded 43 (97.7%) PFIT-s scores at hospital discharge and 37 (86.0%) of these assessments were blinded., Discussion: Our pilot RCT suggests that a future multicentre RCT of early in-bed cycling for MV patients in the ICU is feasible., Trial Registration Number: NCT02377830., Competing Interests: Competing interests: Restorative Therapies (Baltimore, MD) provided 2 RT-300 supine cycle ergometers for Toronto General Hospital and London Health Sciences sites for this research.

A panel of iridium(iii) porphyrin complexes containing axial N-heterocyclic carbene (NHC) ligand(s) were synthesized and characterized. X-ray crystal structures of the bis-NHC complexes [Ir III (ttp)(IMe) 2 ] + ( 2a ), [Ir III (oep)(BIMe) 2 ] + ( 2d ), [Ir III (oep)(I i Pr) 2 ] + ( 2e ) and [Ir III (F 20 tpp)(IMe) 2 ] + ( 2f ) display ruffled porphyrin rings with mesocarbon displacements of 0.483-0.594 Å and long Ir-C NHC bonds of 2.100-2.152 Å. Variable-temperature 1 H NMR analysis of 2a reveals that the macrocycle porphyrin ring inversion takes place in solution with an activation barrier of 40 ± 1 kJ mol -1 . The UV-vis absorption spectra of Ir III (por)-NHC complexes display split Soret bands. TD-DFT calculations and resonance Raman experiments show that the higher-energy Soret band is derived from the 1 MLCT dπ(Ir) → π*(por) transition. The near-infrared phosphorescence of Ir III (por)-NHC complexes from the porphyrin-based 3 (π, π*) state features broad emission bands at 701-754 nm with low emission quantum yields and short lifetimes ( Φ em < 0.01; τ < 4 μs). [Ir III (por)(IMe) 2 ] + complexes (por = ttp and oep) are efficient photosensitizers for 1 O 2 generation ( Φ so = 0.64 and 0.88) and are catalytically active in the light-induced aerobic oxidation of secondary amines and arylboronic acid. The bis-NHC complexes exhibit potent dark cytotoxicity towards a panel of cancer cells with IC 50 values at submicromolar levels. The cytotoxicity of these complexes could be further enhanced upon light irradiation with IC 50 values as low as nanomolar levels in association with the light-induced generation of reactive oxygen species (ROS). Bioimaging of [Ir III (oep)(IMe) 2 ] + ( 2c ) treated cells indicates that this Ir complex mainly targets the endoplasmic reticulum. [Ir III (oep)(IMe) 2 ] + catalyzes the photoinduced generation of singlet oxygen and triggers protein oxidation, cell cycle arrest, apoptosis and the inhibition of angiogenesis. It also causes pronounced photoinduced inhibition of tumor growth in a mouse model of human cancer.