The microenvironment of ovarian follicles in fertile dairy cows is associated with high oocyte quality.

We hypothesised that heifers and cows with positive genetic merit for fertility would have a follicular microenvironment that resulted in better quality oocytes. To test this, we compared cumulus cell-oocyte complexes (COC) and follicular fluid from preovulatory follicles of 36 Holstein-Friesian nulliparous heifers and 50 primiparous lactating cows with either positive (POS, +5%) or negative (NEG, -5%) fertility breeding values (FertBV). Established gene markers of oocyte quality were measured in individual cumulus cell masses and oocytes, and concentrations of amino acids, steroids, and metabolites were quantified in corresponding follicular fluid and plasma. The timing of visually detectable oestrus in NEG FertBV heifers was inconsistent with their stage of COC maturation. Retrospective analyses of oestrous activity data indicated that NEG FertBV heifers were sampled earlier. Their recovered COC were morphologically less mature and exhibited differential expression of genes that are associated with follicular maturation (lower levels of BMPR2) and protein processing (higher levels of HSP90B1). Despite consistent sampling times being achieved in the lactating cows, lower concentrations of serine, proline, methionine, isoleucine, and non-esterified fatty acids were present in follicular fluid from POS FertBV cows. This was associated with higher expression of gene biomarkers of good oocyte quality (VCAN, PDE8A) in COC recovered from POS FertBV cows. This study supports our hypothesis that the follicular microenvironment in lactating dairy cows with high genetic merit leads to COC with higher metabolic rates and oocytes of superior quality. Moreover, an additional stressor such as lactation is required for this difference to be pronounced.

Detection of SARS-CoV-2 within the healthcare environment: a multi-centre study conducted during the first wave of the COVID-19 outbreak in England.

BACKGROUND: Understanding how severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is spread within the hospital setting is essential in order to protect staff, implement effective infection control measures, and prevent nosocomial transmission. METHODS: The presence of SARS-CoV-2 in the air and on environmental surfaces around hospitalized patients, with and without respiratory symptoms, was investigated. Environmental sampling was undertaken within eight hospitals in England during the first wave of the coronavirus disease 2019 outbreak. Samples were analysed using reverse transcription polymerase chain reaction (PCR) and virus isolation assays. FINDINGS: SARS-CoV-2 RNA was detected on 30 (8.9%) of 336 environmental surfaces. Cycle threshold values ranged from 28.8 to 39.1, equating to 2.2 x 105 to 59 genomic copies/swab. Concomitant bacterial counts were low, suggesting that the cleaning performed by nursing and domestic staff across all eight hospitals was effective. SARS-CoV-2 RNA was detected in four of 55 air samples taken <1 m from four different patients. In all cases, the concentration of viral RNA was low and ranged from <10 to 460 genomic copies/m3 air. Infectious virus was not recovered from any of the PCR-positive samples analysed. CONCLUSIONS: Effective cleaning can reduce the risk of fomite (contact) transmission, but some surface types may facilitate the survival, persistence and/or dispersal of SARS-CoV-2. The presence of low or undetectable concentrations of viral RNA in the air supports current guidance on the use of specific personal protective equipment for aerosol-generating and non-aerosol-generating procedures.

Rationale for a reverse tapered body shift implant for immediate placement.

Immediate implant placement holds considerable value, yet primary implant stability is often a critical factor. The aim of this study was to evaluate the stability, volumetric viability, and buccal gap size of reverse tapered body shift (RTBS) implants after immediate placement. Peak insertion torque measurements of two RTBS designs (apical 40% vs. apical 50%), relative to conventionally tapered implants, were assessed in simulated extraction sockets prepared in synthetic bone blocks. Additionally, the proximity of the RTBS implants to neighbouring teeth and anatomical structures, and the buccal gap distance were evaluated in human cadavers. The mean (± standard deviation) insertion torque was 12.00±1.40N•cm for the conventionally tapered implants (n=50), 35.36±2.74N•cm (n=50) for RTBS-1, and 48.20±2.90N•cm (n=50) for RTBS-2; the difference between designs was statistically significant (P<0.01). In total, 40 RTBS implants (20 per design) were placed in six cadaveric premaxillae. Only one locus was inappropriate for both RTBS implant designs, due to the proximity of neighbouring teeth. The average buccal gap for both implant designs was 2.8mm (P=0.104). The improved primary stability and increased buccal gap size with RTBS implants may enhance the feasibility of immediate placement. The study findings should be further validated in clinical trials.

Optimizing Patient Education of Oncology Medications: A Patient Perspective.

The medication information needs of patients with cancer have been primarily studied using quantitative methods and little qualitative research on this topic exists. The purpose of this study was to explore patients' perspectives of optimal oncology medication education provided to patients at the Nova Scotia Health Authority (NSHA). Adult (≥ 18 years) outpatients in medical, gynecological and hematology oncology at NSHA were invited to participate in focus groups, which were audio-recorded, transcribed and analyzed thematically. Three focus groups, including 21 outpatients, were conducted. Four major themes were identified: (1) preparing for what lies ahead consisted of: readiness to receive information, anxiety over the unknown, setting expectations and patients supporting one another; (2) bridging the information gaps was made up of gap in provision of patient education, gap in continuity of patient education, and gap in trustworthy information; (3) understanding the education needs of the patients was comprised of sources of information, education timing and setting, prioritizing information needs, and individuality; and (4) experience within the health care system encompassed: interactions with health care professionals, willingness to ask questions, patient satisfaction, and financial implications. This study identified previously unknown patient education needs and also supported ideas reported in the literature. This data will guide the strategies that will be used to optimize the delivery of oncology medication education at our facility and other health care institutions.

The effect of a non-denaturing detergent and a guanidinium-based inactivation agent on the viability of Ebola virus in mock clinical serum samples.

The 2014 Ebola outbreak in West Africa required the rapid testing of clinical material for the presence of potentially high titre Ebola virus (EBOV). Safe, fast and effective methods for the inactivation of such clinical samples are required so that rapid diagnostic tests including downstream analysis by RT-qPCR or nucleotide sequencing can be carried out. One of the most commonly used guanidinium - based denaturing agents, AVL (Qiagen) has been shown to fully inactivate EBOV once ethanol is added, however this is not compatible with the use of automated nucleic acid extraction systems. Additional inactivation agents need to be identified that can be used in automated systems. A candidate inactivation agent is Triton X-100, a non-denaturing detergent that is frequently used in clinical nucleic acid extraction procedures and has previously been used for inactivation of EBOV. In this study the effect of 0.1% and 1.0% Triton X-100 (final concentration 0.08% and 0.8% respectively) alone and in combination with AVL on the viability of EBOV (106 TCID50/ml) spiked into commercially available pooled negative human serum was tested. The presence of viable EBOV in the treated samples was assessed by carrying out three serial passages of the samples in Vero E6 cells (37°C, 5% CO2, 1 week for each passage). At the end of each passage the cells were observed for evidence of cytopathic effect and samples were taken for rRT-PCR analysis for the presence of EBOV RNA. Before cell culture cytotoxic components of AVL and Triton X-100 were removed from the samples using size exclusion spin column technology or a hydrophobic adsorbent resin. The results of this study showed that EBOV spiked into human serum was not fully inactivated when treated with either 0.1% (v/v) Triton X-100 for 10 mins or 1.0% (v/v) Triton X-100 for 20 mins (final concentrations 0.08% and 0.8% Triton X-100 respectively). AVL alone also did not consistently provide complete inactivation. Samples treated with both AVL and 0.1% Triton X-100 for 10 or 20 mins were shown to be completely inactivated. This treatment is compatible with downstream analysis by RT-qPCR and next generation sequencing.

Geographic distribution of blood collections in Haiti before and after the 2010 earthquake.

BACKGROUND: The January 2010 Haiti earthquake destroyed the National Blood Transfusion Center and reduced monthly national blood collections by > 46%. Efforts to rapidly scale-up blood collections outside of the earthquake-affected region were investigated. STUDY DESIGN AND METHODS: Blood collection data for 2004-2014 from Haiti's 10 administrative departments were grouped into four regions: Northern, Central, Port-au-Prince and Southern. Analyses compared regional collection totals during the study period. RESULTS: Collections in Port-au-Prince accounted for 52% of Haiti's blood supply in 2009, but fell 96% in February 2010. Haiti subsequently increased blood collections in the North, Central and Southern regions to compensate. By May 2010, national blood collections were only 10·9% lower than in May 2009, with 70% of collections coming from outside of Port-au-Prince. By 2013 national collections (27 478 units) had surpassed 2009 levels by 30%, and Port-au-Prince collections had recovered (from 11 074 units in 2009 to 11 670 units in 2013). CONCLUSION: Haiti's National Blood Safety Program managed a rapid expansion of collections outside of Port-au-Prince following the earthquake. Annual collections exceeded pre-earthquake levels by 2012 and continued rising annually. Increased regional collections provided a greater share of the national blood supply, reducing dependence on Port-au-Prince for collections.

Prenatal diagnosis of trisomy 21, 18 and 13 by quantitative pyrosequencing of segmental duplications.

Chromosomal aberration mostly occurs in chromosomes 21, 18 and 13, with an incidence approximately 1 out of 160 live births in humans, therefore making prenatal diagnosis necessary in clinics. Current methods have drawbacks such as time consuming, high cost, complicated operations and low sensitivity. In this paper, a novel method for rapid and accurate prenatal diagnosis of aneuploidy is proposed based on pyrosequencing, which quantitatively detects the peak height ratio (PHR) of different bases of segmental duplication. A direct polymerase chain reaction (PCR) approach was undertaken, where a small volume of amniotic fluid was used as the starting material without DNA extraction. Single-stranded DNA was prepared from PCR products and subsequently analyzed using pyrosequencing. The PHR between target and reference chromosome of 2.2 for euploid and 3:2 for a trisomy fetus were used as reference. The reference intervals and z scores were calculated for discrimination of aneuploidy. A total of 132 samples were collected, within trisomy 21 (n = 11), trisomy 18 (n = 3), trisomy 13 (n = 2), and unaffected controls (n = 116). A set of six segmental duplications were chosen for analysis. This method had consistent results with karyotyping analysis, a correct diagnosis with 100% sensitivity and 99.9% specificity.

Efficacy of liposome-encapsulated ciprofloxacin in a murine model of Q fever.

Encapsulation of antibiotics may improve treatment of intracellular infections by prolonging antibiotic release and improving antibiotic uptake into cells. In this study, liposome-encapsulated ciprofloxacin for inhalation (CFI) was evaluated as a postexposure therapeutic for the treatment of Coxiella burnetii, the causative agent of Q fever. Intranasal treatment of male A/Jola (A/J) mice with CFI (50 mg/kg of body weight) once daily for 7 days protected mice against weight loss and clinical signs following an aerosol challenge with C. burnetii. In comparison, mice treated twice daily with oral ciprofloxacin or doxycycline (50 mg/kg) or phosphate-buffered saline (PBS) lost 15 to 20% body weight and exhibited ruffled fur, arched backs, and dehydration. Mice were culled at day 14 postchallenge. The weights and bacterial burdens of organs were determined. Mice treated with CFI exhibited reduced splenomegaly and reduced bacterial numbers in the lungs and spleen compared to mice treated with oral ciprofloxacin or doxycycline. When a single dose of CFI was administered, it provided better protection against body weight loss than 7 days of treatment with oral doxycycline, the current antibiotic of choice to treat Q fever. These data suggest that CFI has potential as a superior antibiotic to treat Q fever.

Investments in blood safety improve the availability of blood to underserved areas in a sub-Saharan African country.

BACKGROUND AND OBJECTIVES: Since 2004, several African countries, including Namibia, have received assistance from the U.S. President's Emergency Plan for AIDS Relief (PEPFAR). Gains have been documented in the safety and number of collected units in these countries, but the distribution of blood has not been described. MATERIALS AND METHODS: Nine years of data on blood requests and issues from Namibia were stratified by region to describe temporal and spatial changes in the number and type of blood components issued to Namibian healthcare facilities nationally. RESULTS: Between 2004 and 2007 (early years of PEPFAR support) and 2008-2011 (peak years of PEPFAR support), the average number of red cell units issued annually increased by 23.5% in seven densely populated but less-developed regions in northern Namibia; by 30% in two regions with urban centres; and by 35.1% in four sparsely populated rural regions. CONCLUSION: Investments in blood safety and a policy decision to emphasize distribution of blood to underserved regions improved blood availability in remote rural areas and increased the proportion of units distributed as components. However, disparities persist in the distribution of blood between Namibia's urban and rural regions.

Ranking and selection of MII oocytes in human ICSI cycles using gene expression levels from associated cumulus cells.

STUDY QUESTION: Can the ranked expression levels of a cohort of cumulus cell (CC) genes be used to select MII oocytes with a potential for blastocyst development and live birth? SUMMARY ANSWER: A ranking method containing four (HAS2, FSHR, VCAN, PR) of the eight genes evaluated in this study for identifying good quality MII oocytes provides a significantly better outcome compared with random selection and is equally as good as using all oocytes for ICSI. WHAT IS KNOWN ALREADY: Recent evidence has identified a number of candidate genes in CC that have the potential to serve as markers of oocyte quality; however, a reliable method for selecting MII oocytes with blastocyst and live birth potential remains a challenge. STUDY DESIGN, SIZE, DURATION: A group of 25 patients (<38 years old) underwent rFSH-stimulated ICSI treatment with single embryo replacement (SET). A total of 270 cumulus cell-oocyte complexes (COCs) were recovered and assessed. MATERIALS, SETTING, METHODS: Expression levels of eight candidate genes (HAS2, FSHR, SLC2A4, ALCAM, SFRP2, VCAN, NRP1 and PR), corrected for RPL19, were measured in individual CC masses using multiplex QPCR. Expression levels of individual CC masses were assessed and ranked in relation to oocyte developmental indicators (blastocyst formation and live birth). MAIN RESULTS AND THE ROLE OF CHANCE: From the 25 women, 19 (76%) had achieved a successful live birth delivery following SET. In this population, the selection of MII oocytes according to relative ranking levels of a subset of CC-expressed genes provided a significantly higher chance of identifying a good quality oocyte compared with selecting MII oocytes randomly (blastocyst: 1× MII oocyte: 52 versus 23%, P = 0.008; 3× MII oocytes: 80 versus 52%, P = 0.002; live birth: 1× MII oocyte: 31 versus 15%, P<0.05, 3× MII oocytes: 60 versus 38%, P < 0.05) and a similar chance to that of using all oocytes available after recovery (blastocyst: 80% versus 96%, P = 0.085, live birth: 60% versus 76%, P = 0.206). LIMITATIONS, REASONS FOR CAUTION: The present method was validated only for young (<38 years) women, with male infertility, who had no signs of androgenicity, PCOS or endometriosis and were free of any chronic disease. This is a retrospective study that requires further validation in an unselected population. WIDER IMPLICATIONS OF THE FINDINGS: Results presented in this study could be used to assist the selection of oocytes with high blastocyst developmental potential in frozen oocyte cycles and for the selection of embryos with high developmental potential as early as 18 h after ICSI (2PN stage) in fresh human IVF cycles. STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by Fertility Associates Ltd and the New Zealand Government. The authors declare there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Reduced risk of transfusion-transmitted HIV in Kenya through centrally co-ordinated blood centres, stringent donor selection and effective p24 antigen-HIV antibody screening.

BACKGROUND: Following a 1994 study showing a high rate of transfusion-associated HIV, Kenya implemented WHO blood safety recommendations including: organizing the Kenya National Blood Transfusion Service (NBTS), stringent blood donor selection, and universal screening with fourth-generation p24 antigen and HIV antibody assays. Here, we estimate the risk of transfusion-associated HIV transmission in Kenya resulting from NBTS laboratory error and consider the potential safety benefit of instituting pooled nucleic acid testing (NAT) to reduce window period transmission. METHODS: From November to December 2008 in one NBTS regional centre, and from March to June 2009 in all six NBTS regional centres, every third unit of blood screened negative for HIV by the national algorithm was selected. Dried blood spots were prepared and sent to a reference laboratory for further testing, including NAT. Test results from the reference laboratory and NBTS were compared. Risk of transfusion-associated HIV transmission owing to laboratory error and the estimated yield of implementing NAT were calculated. FINDINGS: No cases of laboratory error were detected in 12,435 units tested. We estimate that during the study period, the percentage of units reactive for HIV by NAT but non-reactive by the national algorithm was 0·0% (95% exact binomial confidence interval, 0·00-0·024%). INTERPRETATION: By adopting WHO blood safety strategies for resource-limited settings, Kenya has substantially reduced the risk of transfusion-associated HIV infection. As the national testing and donor selection algorithm is effective, implementing NAT is unlikely to add a significant safety benefit. These findings should encourage other countries in the region to fully adopt the WHO strategies.

At what cost care?

This paper looks at the concept of care in nursing and considers the ever-changing focus relative to the meaning of the term care and how this care is delivered by nursing staff. In the process of looking at these issues it examines the theoretical and practical issues and how these factors have changed considerably over the last twenty-five years. This examination touches upon some of the intermingled and not mutually exclusive issues which surround care and caring such as technology, stress and burnout, bureaucracy, fiscal policy, the humanness of the nurse and the ever changing nature of care delivery. Many readers will be able to relate to the issues discussed and understand how some of these factors tend to get in the way of one another and affect good patient care and outcomes.

Normal reproductive and macrophage function in Pem homeobox gene-deficient mice.

Interaction between germ cells and the supporting somatic cells guides many of the differentiative processes of gametogenesis. The expression pattern of the Pem homeobox gene suggests that it may mediate specific inductive events in murine reproductive tissues. During gestation, Pem is expressed in migrating and early postmigratory primordial germ cells, as well as in all embryo-derived extraembryonic membranes. Pem expression ceases in the germline after Embryonic Day 14 in both sexes and then reappears postnatally in the supporting cells of the gonad. In mature mice, Pem is produced by testicular Sertoli cells during stages VI-VIII of spermatogenesis and transiently by ovarian granulosa cells lining periovulatory follicles. Despite this tightly regulated reproductive expression pattern, mice with a targeted mutation in Pem have normal fecundity, with no detectable alteration in extraembryonic testicular or ovarian development or function. We also show that Pem is expressed throughout embryonic and adult development in a subset of a tissue-specific class of macrophages, Kupffer cells, as well as in a localized fraction of cells in macrophage cell lines. Although the number of Pem-positive Kupffer cells increases in mice treated with lipopolysaccharide, loss of Pem does not detectably interfere with the cells' ability to induce iNOS expression, demonstrating this Kupffer cell function does not require Pem. No differences were observed between Pem-knockout mice in 129, C57BL6/J, or mixed genetic backgrounds. Together, these data show that Pem is dispensable for embryonic and postnatal development, gonadal function, and Kupffer cell activation, perhaps due to compensatory expression of a similar homeobox gene.

The identification of secreted heat shock 60 -like protein from rat glial cells and a human neuroblastoma cell line.

The intracellular stress-induced proteins provide protection against toxic insults. Here, a 60,000-Da heat shock 60 (hsp60)-like protein was detected, with five different antibodies, in conditioned media derived from rat cortical astrocytes and a human neuroblastoma cell line. Extracellular neuroblastoma hsp60-like immunoreactivity was increased 3-fold in the presence of the neuropeptide vasoactive intestinal peptide (VIP) and was augmented 2-fold after temperature elevation. Intracellular hsp60 immunoreactivity was reduced 2-3-fold in the presence of VIP; this reduction was attenuated in the presence of brefeldin A, an inhibitor of protein secretion. In contrast, the activity of lactate dehydrogenase (LDH), an intracellular marker, did not change in the presence of VIP. Essentially no extracellular LDH activity was detected, indicating no cellular damage. A novel aspect for stress proteins having extracellular protective roles is suggested.

The Pem homeobox gene is X-linked and exclusively expressed in extraembryonic tissues during early murine development.

We previously reported the isolation of a cDNA clone for a homeobox-containing gene designated Pem, shown by Northern analysis of Day 7 through Day 16 mouse embryos to be expressed in extraembryonic tissues. In this study, Pem gene expression was further examined using in situ hybridization and immunocytochemistry to determine the spatial distribution of Pem transcripts and protein in peri-implantation embryos and in embryoid bodies (EBs). Low amounts of Pem mRNA were detected in undifferentiated EBs. When EBs were induced to differentiate, the outer cell layer of visceral or parietal endoderm expressed both Pem mRNA and protein. In developing embryos, no Pem protein was detectable in the uncompacted morula; 12% of the nuclei in compacted morulae were Pem positive, while 25% of the blastocyst trophectoderm and 15% of inner cell mass cells expressed Pem protein. Shortly after implantation, in 5.5 and 6.5 d.p.c. embryos, Pem expression was limited to extraembryonic tissues and was present in distal and proximal visceral endoderm, parietal endoderm, and ectoplacental cone. By 7.5-8.5 d.p.c. neither Pem RNA nor protein was found in the distal squamous visceral endoderm, which surrounds the embryonic region of the egg cylinder, nor in the parietal endoderm. Expression was retained in the proximal columnar epithelium of the visceral endoderm. Prominent Pem expression was observed in the chorion, in trophoblast-derived cells of the ectoplacental cone, and in secondary giant cells, localized in the nuclear compartment. Pem was localized to the X chromosome and found to be expressed in cell lineages where only the maternal X chromosome is active. The data indicate a possible role for Pem in regulating genes involved in the differentiation of extraembryonic tissues.

The time course and location of cerebral evoked activity associated with the processing of colour stimuli in man.

Area V4 has been located in man in the region of the fusiform gyrus on the inferior surface of the occipital lobe. Using multiple dipole source analysis on multichannel EEG recordings of visual evoked potentials to coloured 'Mondrian' stimuli in man, we have confirmed that activity is consistently seen in this area regardless of the retinal area stimulated and have obtained new information concerning its time course. Three different localized centres of activity follow the visual stimulus, with peak latencies of 90, 110 and 160 ms, and arising respectively in the region of visual areas V1, V2/V3 and V4. The time course and character of the V4 dipole activity to a colourless black-and-white Mondrian is indistinguishable from that to the coloured Mondrian, supporting the evidence that the cells of V4 are not exclusively concerned with colour processing.