Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases.

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.

Pathogen invasion-dependent tissue reservoirs and plasmid-encoded antibiotic degradation boost plasmid spread in the gut.

Many plasmids encode antibiotic resistance genes. Through conjugation, plasmids can be rapidly disseminated. Previous work identified gut luminal donor/recipient blooms and tissue-lodged plasmid-bearing persister cells of the enteric pathogen Salmonella enterica serovar Typhimurium (S.Tm) that survive antibiotic therapy in host tissues, as factors promoting plasmid dissemination among Enterobacteriaceae. However, the buildup of tissue reservoirs and their contribution to plasmid spread await experimental demonstration. Here, we asked if re-seeding-plasmid acquisition-invasion cycles by S.Tm could serve to diversify tissue-lodged plasmid reservoirs, and thereby promote plasmid spread. Starting with intraperitoneal mouse infections, we demonstrate that S.Tm cells re-seeding the gut lumen initiate clonal expansion. Extended spectrum beta-lactamase (ESBL) plasmid-encoded gut luminal antibiotic degradation by donors can foster recipient survival under beta-lactam antibiotic treatment, enhancing transconjugant formation upon re-seeding. S.Tm transconjugants can subsequently re-enter host tissues introducing the new plasmid into the tissue-lodged reservoir. Population dynamics analyses pinpoint recipient migration into the gut lumen as rate-limiting for plasmid transfer dynamics in our model. Priority effects may be a limiting factor for reservoir formation in host tissues. Overall, our proof-of-principle data indicates that luminal antibiotic degradation and shuttling between the gut lumen and tissue-resident reservoirs can promote the accumulation and spread of plasmids within a host over time.

Steep Declines in Pediatric AIDS Mortality in South Africa, Despite Poor Progress Toward Pediatric Diagnosis and Treatment Targets.

BACKGROUND: Few attempts have been made to monitor progress toward HIV diagnosis and antiretroviral treatment (ART) coverage targets in children, and the impact that ART and prevention of mother-to-child transmission (PMTCT) programs have had on pediatric HIV incidence and mortality. METHODS: A multiparameter evidence synthesis approach was adopted to integrate South African pediatric HIV data sources. A previously developed model of HIV in South Africa was calibrated to household survey HIV prevalence data, routine antibody testing data, data on numbers and ages of children on ART, vital registration data and data on HIV diagnosis at death. The impact of ART and PMTCT was estimated by comparing validated model outputs against model predictions of the trends that would have been expected in the absence of ART and PMTCT. RESULTS: By mid-2018, the model estimated that 75.2% (95% CI: 73.9%-76.8%) of HIV-positive children were diagnosed, substantially lower than the corresponding estimates in HIV-positive adults (91.0%). ART coverage in children in 2018 (51.2%, 95% CI: 49.4%-52.7%) was also lower than that in adults (62.0%). In 2017-2018, the numbers of new cases of mother-to-child transmission and pediatric AIDS deaths were reduced by 84% and 94%, respectively, relative to what would have been expected in the absence of interventions, but reductions in mortality were driven largely by PMTCT. CONCLUSIONS: Although levels of AIDS mortality in children have declined dramatically in South Africa, this has mostly been due to successful PMTCT programs, and progress toward the 90-90-90 targets appears relatively poor when compared with that in adults.

Microfluidic Multitissue Platform for Advanced Embryotoxicity Testing In Vitro.

The integration of metabolic competence in developmental toxicity assays in vitro is of fundamental importance to better predict adverse drug effects. Here, a microfluidic hanging-drop platform is presented that seamlessly integrates liver metabolism into the embryonic stem cell test (EST). Primary human liver microtissues (hLiMTs) and embryoid bodies (EBs) are combined in the same fluidic network, so that hLiMT-generated metabolites are directly transported to the EBs. Gravity-driven flow through the network enables continuous intertissue communication, constant medium turnover, and, most importantly, immediate exchange of metabolites. As a proof of concept, the prodrug cyclophosphamide is investigated and a fourfold lower ID50 concentration (50% inhibition of EB differentiation) is found after biotransformation, which demonstrates the potentially adverse effects of metabolites on embryotoxicity. The metaEST platform provides a promising tool to increase the predictive power of the current EST assay by more comprehensively including and better reflecting physiological processes in in vitro tests.

Delay and poor diagnosis of Down syndrome in KwaZulu-Natal, South Africa: A retrospective review of postnatal cytogenetic testing.

BACKGROUND: Down syndrome (DS) is the most common chromosomal disorder in newborns. Until 20 years ago DS was considered rare in black African children in South Africa (SA). Lack of awareness of DS on the part of medical staff in SA, and difficulty in diagnosing it, appear to persist. OBJECTIVES: To establish an epidemiological profile of DS and investigate the ability of clinicians in KwaZulu-Natal Province (KZN), SA, to make accurate clinical diagnoses of DS. METHODS: Records at the South African National Blood Service cytogenetic laboratory in Pinetown, KZN, were examined for all tests for clinically suspected DS undertaken during January 2009 - December 2013 and all cytogenetically proven DS test results. Age at diagnosis, the hospital from where the test was sent and type of chromosomal pattern for each confirmed DS test result were recorded. RESULTS: Of a total of 1 578 tests requested, 875 confirmed DS, indicating that clinicians correctly clinically diagnosed DS 55.4% of the time. The average age of cytogenetic diagnosis of DS was 1 year and 20 days. The minimum population prevalence of DS was 0.8/1 000. CONCLUSIONS: The diagnosis of DS is a challenge in KZN, potentiating missed opportunities for early intervention. The relatively low population prevalence of DS may be attributable to a lack of confirmatory cytogenetic tests or missed clinical diagnoses. It may also be attributable to a high mortality rate for children with DS in the province.

Seamless Combination of Fluorescence-Activated Cell Sorting and Hanging-Drop Networks for Individual Handling and Culturing of Stem Cells and Microtissue Spheroids.

Open microfluidic cell culturing devices offer new possibilities to simplify loading, culturing, and harvesting of individual cells or microtissues due to the fact that liquids and cells/microtissues are directly accessible. We present a complete workflow for microfluidic handling and culturing of individual cells and microtissue spheroids, which is based on the hanging-drop network concept: The open microfluidic devices are seamlessly combined with fluorescence-activated cell sorting (FACS), so that individual cells, including stem cells, can be directly sorted into specified culturing compartments in a fully automated way and at high accuracy. Moreover, already assembled microtissue spheroids can be loaded into the microfluidic structures by using a conventional pipet. Cell and microtissue culturing is then performed in hanging drops under controlled perfusion. On-chip drop size control measures were applied to stabilize the system. Cells and microtissue spheroids can be retrieved from the chip by using a parallelized transfer method. The presented methodology holds great promise for combinatorial screening of stem-cell and multicellular-spheroid cultures.

Range resolved mode mixing in a large volume for the mitigation of speckle and strategic target orientation requirements in active millimeter-wave imaging.

In spite of many reports of active millimeter-wave imaging in the literature, speckle and requirements for cooperative target orientation significantly reduce its practical usefulness. Here we report a new technique, range resolved mode mixing (RRMM), which significantly mitigates both of these issues. It also provides a three-dimensional (3D) image. RRMM accomplishes this by combining multimode illumination (which eliminates the requirement for cooperative target orientation) with range resolution (which provides statistical independence of speckle patterns for averaging and the 3D image). The use of a 5W extended interaction klystron amplifier results in large signal margins in the 50 m scale atrium of the Physics Department at Ohio State University. It appears that there are a number of scenarios out to a range of 1 km for which this approach is useful to provide 3D images, with minimal speckle, and no requirement for cooperative target orientation.

Multimode illumination for speckle reduction and angle neutrality in millimeter wave active imaging: range and time-resolved mode averaging.

Active illumination is an attractive approach for millimeter and submillimeter wave imaging because of its generally larger signal margins and capacity for range determination. However, the resultant speckle from diffuse targets and the requirement that specular targets have strategic angular alignment are significant shortcomings. As a result, many, if not most, demonstrations of active imaging have involved the use of strategically oriented specular targets. We have previously shown [J. Opt. Soc. Am. A29, 2643 (2012)10.1364/JOSAA.29.002643JOAOD61084-7529] that spatially time-resolved modulated multimode mixing approaches mitigate these issues. In this paper, we extend this work to demonstrate the use of range resolved multimode mixing as an alternative means to provide statistically independent images for speckle reduction averaging. We also demonstrate the use of systematic illumination of spatial modes in a "grassy field" geometry.

Improving paediatric tuberculosis and HIV clinical record keeping: the use of audit and a structured pro forma in a South African regional level hospital.

We report on the impact of revisions made to an existing pro forma facilitating routine assessment and the management of paediatric HIV and tuberculosis (TB) in KwaZulu-Natal, South Africa. An initial documentation audit in 2010 assessed 25 sets of case notes for the documentation of 16 select indicators based on national HIV and TB guidelines. Using the findings of this initial audit, the existing case note pro forma was revised. The introduction of the revised pro forma was accompanied by training and a similar repeat audit was undertaken in 2012. This demonstrated an overall improvement in documentation. The three indicators that improved most were documentation of maternal HIV status, child's HIV status and child's TB risk assessment (all P < 0.001). This study suggests that tailor-made documentation pro formas may have an important role to play in improving record keeping in low-resource settings.

Elimination of speckle and target orientation requirements in millimeter-wave active imaging by modulated multimode mixing illumination.

Active imaging can provide significantly larger signal margins in the millimeter-wave spectral region than passive imaging, especially indoors-an important application for which there is no cold sky illumination. However, coherent effects, such as speckle, negate much of this advantage by destroying image clarity and target recognition. Moreover, active imaging demonstrations often use strategically chosen target orientations to optimally reflect power from the active illuminator back to the imaging receiver. In this paper we will discuss and show experimental results for a new active imaging approach that largely eliminates coherent effects and the need for optimized target orientation. The work described uses a synthesized harmonic multiplier chain to drive a 5 W extended interaction klystron at 218.4 GHz, a mechanical mode mixer to illuminate and modulate many modes, and a heterodyne receiver coupled into a 60 cm scanning mirror. Large signal margins were obtained in this ~50 m range work, showing paths to imaging at ~1 km, imaging with considerably less powerful illuminators, and the use of focal plane arrays.

Optimization of a series of 4,6-bis-anilino-1H-pyrrolo[2,3-d]pyrimidine inhibitors of IGF-1R: elimination of an acid-mediated decomposition pathway.

Initial evaluation of a series 4,6-bis-anilino-1H-pyrrolo[2,3-d]pyrimidines revealed a C(1') carboxamide was preferred for sub-micromolar in vitro potency against IGF-1R. Subsequent solution stability studies with 1 revealed a susceptibility toward acid-induced intramolecular cyclization with the C(1') carboxamide. Herein, we describe several successful approaches toward generating both potent and acid-stable inhibitors of IGF-1R within the 4,6-bis-anilino-1H-pyrrolo[2,3-d]pyrimidine template.

Every death counts: use of mortality audit data for decision making to save the lives of mothers, babies, and children in South Africa.

South Africa is one of the few developing countries with a national confidential inquiry into maternal deaths. 164 health facilities obtain audit data for stillbirths and neonatal deaths, and a new audit network does so for child deaths. Three separate reports have been published, providing valuable information about avoidable causes of death for mothers, babies, and children. These reports make health-system recommendations, many of which overlap and are intertwined with the scarcity of progress in addressing HIV/AIDS. The leaders of these three reports have united to prioritise actions to save the lives of South Africa's mothers, babies, and children. The country is off-track for the health-related Millennium Development Goals. Mortality in children younger than 5 years has increased, whereas maternal and neonatal mortality remain constant. This situation indicates the challenge of strengthening the health system because of high inequity and HIV/AIDS. Coverage of services is fairly high, but addressing the gaps in quality and equity is essential to increasing the number of lives saved. Consistent leadership and accountability to address crosscutting health system and equity issues, and to prevent mother-to-child transmission of HIV, would save tens of thousands of lives every year. Audit is powerful, but only if the data lead to action.

Childhood death auditing to improve paediatric care.

AIM: Field-testing a paediatric mortality audit system at eight sites in South Africa to assess its feasibility, to collect local data on common causes of death in children under 5 y, and to determine health system failure, missed opportunities of intervention and substandard care (modifiable factors). METHODS: The study tested the Under-5 Healthcare Problem Identification Programme (U5PIP). The sites represent different levels of paediatric healthcare. STUDY PERIOD: 1 September 2003-31 August 2004. STUDY POPULATION: Under-5 patients admitted to study hospitals. CASES: Under-5 patients who died in study hospitals. RESULTS: In total, 1532 under-5 deaths occurred, representing a case-fatality rate of 7.8%. Main causes of death were lower respiratory tract infections (33%), gastroenteritis (15%) and septicaemia (12%). Sixty per cent of the deaths were HIV/AIDS related. Sixty-nine per cent of children who died were underweight. Administrative modifiable factors were present in 31% of deaths. Clinical personnel-related modifiable factors were detected in 26% at the clinic level and in 33-37% at the hospital level. CONCLUSION: The U5PIP is feasible for ongoing mortality reviews by paediatric teams as part of routine work. Information on common causes of death and modifiable factors in this study focus on the impact of HIV/AIDS, malnutrition and resource allocation, and can be used for interventions to improve paediatric healthcare.

Cough and reflux esophagitis in children: their co-existence and airway cellularity.

BACKGROUND: There are no prospective studies that have examined for chronic cough in children without lung disease but with gastroesophageal reflux (GER). In otherwise healthy children undergoing flexible upper gastrointestinal endoscopy (esophago-gastroscopy), the aims of the study were to (1) define the frequency of cough in relation to symptoms of GER, (2) examine if children with cough and reflux esophagitis (RE) have different airway cellularity and microbiology in bronchoalveolar lavage (BAL) when compared to those without. METHODS: Data specific for chronic cough (> 4-weeks), symptoms of GER and cough severity were collected. Children aged < 16-years (n = 150) were defined as 'coughers' (C+) if a history of cough in association with their GER symptoms was elicited before BAL were obtained during elective esophago-gastroscopy. Presence of esophagitis on esophageal biopsies was considered reflux esophagitis positive (E+). RESULTS: C+ (n = 69) were just as likely as C- (n = 81) to have esophagitis, odds ratio 0.87 (95%CI 0.46, 1.7). Median neutrophil percentage in BAL was significantly different between groups; highest in C+E- (7, IQR 28) and lowest in C-E+ (5, IQR 6). BAL positive bacterial culture occurred in 20.7% and were more likely present in current coughers (OR 3.37, 95%CI 1.39, 8.08). Airway neutrophilia (median 20%, IQR 34) was significantly higher in those with BAL positive bacterial cultures than those without (5%, 4; p = 0.0001). CONCLUSION: In children without lung disease, the common co-existence of cough with symptoms of GER is independent of the occurrence of esophagitis. Airway neutrophilia when present in these children is more likely to be related to airway bacterial infection and not to esophagitis.

Implementation of kangaroo mother care: a randomized trial of two outreach strategies.

AIM: To test whether a well-designed educational package on the implementation of kangaroo mother care (KMC) used on its own can be as effective in implementing KMC in a healthcare facility as the combination of a visiting facilitator used in conjunction with the package. SETTING: Thirty-four hospitals in KwaZulu-Natal Province, South Africa. METHOD: The hospitals were paired with respect to their geographical location and annual number of births at the facility. One hospital in each pair was randomly allocated to receive either the implementation package alone (group A) or the implementation package and visits from a facilitator (group B). Hospitals in group B received three facilitation visits. All hospitals were evaluated by a site visit 8 mo after launching the process and were scored by means of a progress-monitoring tool. OUTCOMES: Successful implementation was regarded as demonstrating evidence of practice (score>10) during the site visit. RESULTS: Group B scored significantly better than group A (p<0.05). All 17 hospitals in group B demonstrated evidence of practice, with the median score of the group being 15.44 (range 10.29-22.94). Twelve of the hospitals in group A demonstrated evidence of practice and the median score was 11.33 (range 1.08-21.13). CONCLUSION: Successful implementation was achieved in most of the hospitals irrespective of the strategy used. However, facilitation with an implementation package was clearly superior to using a package alone. Some sites do not need facilitation for successful implementation.

Measuring implementation progress in kangaroo mother care.

AIM: To describe the development and testing of a monitoring model with quantitative indicators or progress markers that could measure the progress of individual hospitals in the implementation of kangaroo mother care (KMC). METHODS: Three qualitative data sets in the larger research programme on the implementation of KMC of the MRC Research Unit for Maternal and Infant Health Care Strategies in South Africa were used to develop a progress-monitoring model and an accompanying instrument. RESULTS: The model was conceptualized around three phases (pre-implementation, implementation and institutionalization) and six constructs depicting progress (awareness, adopting the concept, mobilization of resources, evidence of practice, evidence of routine and integration, sustainable practice). For each construct, indicators were developed for which data could be collected by means of the monitoring instrument used in a walk-through visit to a hospital. The instrument has been tested in 65 hospitals. CONCLUSION: The progress-monitoring model enables the quantification of individual hospitals' progress in the process of implementing KMC and an objective measurement of the effectiveness of different outreach strategies. The model also has potential to be adapted for measuring progress in other innovative healthcare interventions on a large scale.

Three-dimensional echocardiography is superior to multiplane transoesophageal echo in the assessment of regurgitant mitral valve morphology.

AIMS: Transoesophageal echocardiography (TOE) plays a vital role in the assessment of mitral valve morphology. However, the accuracy of TOE may be limited by inadequate recognition of all segments. We aimed to evaluate the role of three-dimensional (3D) echocardiography in this respect. METHODS AND RESULTS: Seventy-five patients were studied prior to mitral valve repair surgery. A scoring protocol was devised for recognition of the eight Carpentier segments (0=inadequate for analysis, 1=adequate, 2=good). Using surgical findings as the gold standard, TOE and 3D were compared for adequate recognition scores and accurate detection of functional morphology. Adequate recognition was more frequently obtained with 3D imaging (97% of segments by 3D c.f. 90% by TOE; p = 0.000). The major difference was seen at the commissures (adequate scores in 143/150 commissures by 3D c.f. 90/150 by TOE; p = 0.000). 3D matched more closely to surgical findings, achieving exact functional description in 92% of segments vs 79% segments with TOE (p = 0.000). This incremental value of 3D was seen in both commissures and the anterior leaflet but not in the posterior leaflet. CONCLUSIONS: In this study 3D was superior not only for complete recognition of the mitral valve but also for the accurate localisation and identification of pathology.