This protocol describes the automated extraction of HMW DNA from nucleated blood samples intended for long-read sequencing using the Nanobind® tissue kit and the Thermo Fisher KingFisher™ Apex. It follows the "Nanobind® HMW DNA Extraction from 5 μL nucleated red blood cell on KingFisher Apex" R&D protocol, which was developed by Pacific Biosciences and validated by the Tree of Life Core Laboratory using bird, fish and reptile nucleated blood samples. This process is effective for any species with nucleated blood within the chordate group, covered by the Tree of Life Programme, however difficulties can arise with samples where preservation has not been optimal. In addition to allowing a higher throughput of nucleated blood samples, this protocol is also particularly beneficial for nucleated blood samples where clumping has occurred, with higher yields obtained compared to processing them using the Manual Nucleated Blood Nanobind® protocol. The output of this protocol is HMW DNA of high quality and quantity, which can be directed towards the HMW DNA Fragmentation: Diagenode Megaruptor®3 for LI PacBio protocol. Acronyms: HMW: high-molecular weight LI: low input
The potential value of DNA barcoding for the identification of medicinal plants and authentication of traded plant materials has been widely recognized; however, a number of challenges remain before DNA methods are fully accepted as an essential quality control method by industry and regulatory authorities. The successes and limitations of conventional DNA barcoding are considered in relation to important members of the Lamiaceae. The mint family (Lamiaceae) contains over one thousand species recorded as having a medicinal use, with many more exploited in food and cosmetics for their aromatic properties. The family is characterized by a diversity of secondary products, most notably the essential oils (EOs) produced in external glandular structures on the aerial parts of the plant that typify well-known plants of the basil (Ocimum), lavender (Lavandula), mint (Mentha), thyme (Thymus), sage (Salvia) and related genera. This complex, species-rich family includes widely cultivated commercial hybrids and endangered wild-harvested traditional medicines, and examples of potential toxic adulterants within the family are explored in detail. The opportunities provided by next generation sequencing technologies to whole plastome barcoding and nuclear genome sequencing are also discussed with relevant examples.
The prescribing authorities, clinical activities, and productivity documentation strategies of ambulatory care clinic-based pharmacists practicing within a large academic health system are described.North Carolina law encourages progressive pharmacy practice through acquisition of the clinical pharmacist practitioner (CPP) designation. Qualified CPPs are authorized to provide collaborative drug therapy management services, including medication prescribing and ordering of laboratory tests, according to defined protocols and under physician supervision. The University of North Carolina Medical Center has approximately 30 CPPs deployed across a wide range of ambulatory care clinical practice sites. This article describes (1) the pharmacy department's implementation of an ambulatory care practice model, (2) the credentialing and privileging process leading to granting of prescribing privileges, (3) metrics used to demonstrate the impact of CPP activities, (4) recommended general criteria for ambulatory care practice site identification, and (5) strategies for overcoming barriers to successful implementation of ambulatory care-focused clinical pharmacist services. Aggregated intervention-tracking data compiled by seven of the medical center's CPP ambulatory care practice sites indicate extensive CPP involvement in direct patient care encounters and patient or provider consultations, with large numbers of medication-related interventions to support institutional cost-avoidance and revenue goals.CPPs deployed at the medical center's ambulatory care clinics have had a positive impact on clinical and cost outcomes, improving patient care through interventions, contributing to readmission reduction efforts, generating indirect revenue through cost avoidance, and generating new revenue through billing for patient visits.
Tulsi (Holy basil, Ocimum tenuiflorum L., Lamiaceae), native to Asia, has become globalised as the cultural, cosmetic, and medicinal uses of the herb have been popularised. DNA barcoding, a molecular technique used to identify species based on short regions of DNA, can discriminate between different species and identify contaminants and adulterants. This study aimed to explore the values associated with Tulsi in the United Kingdom (UK) and authenticate samples using DNA barcoding. A mixed methods approach was used, incorporating social research (i.e., structured interviews) and DNA barcoding of Ocimum samples using the ITS and trnH-psbA barcode regions. Interviews revealed the cultural significance of Tulsi: including origins, knowledge exchange, religious connotations, and medicinal uses. With migration, sharing of plants and seeds has been seen as Tulsi plants are widely grown in South Asian (SA) households across the UK. Vouchered Ocimum specimens (n = 33) were obtained to create reference DNA barcodes which were not available in databases. A potential species substitution of O. gratissimum instead of O. tenuiflorum amongst SA participants was uncovered. Commercial samples (n = 47) were difficult to authenticate, potentially due to DNA degradation during manufacturing processes. This study highlights the cultural significance of Tulsi, despite a potential species substitution, the plant holds a prestigious place amongst SA families in the UK. DNA barcoding was a reliable way to authenticate Ocimum species.
DNA barcoding is a widely accepted technique for the identification of plant materials, and its application to the authentication of commercial medicinal plants has attracted significant attention. The incorporation of DNA-based technologies into the quality testing protocols of international pharmacopoeias represents a step-change in status, requiring the establishment of standardized, reliable and reproducible methods. The process by which this can be achieved for any herbal medicine is described, using
Since the implementation of NHS Reform and Community Care Act in early 1990s, the structure of mental health services has changed dramatically. This has in turn influenced the changing role of mental health nurses. The mental health nursing review in 1994 further reinforced the need for mental health nurses to be more robust in closing the theory-practice divide in order to develop a profession which is reflective and self-appraising.
The Plant Organic HMW gDNA Extraction (POE) protocol acts as the Sanger Tree of Life's programmes mid-throughput, reserve gDNA extraction procedure for all plant species too recalcitrant to yield HMW gDNA of adequate quality or quantity with the Plant MagAttract v.4 protocol. Developed in-house, the POE protocol is highly efficient at isolating pure, high-quality and high molecular weight (HMW) gDNA from the majority of plant species to an extent adequate for long-read sequencing. The POE protocol is divided into four stages: (1) direct lysis of tissue homogenates with an SDS-based buffer containing reducing agents to mitigate oxidative DNA damage, (2) centrifugation and KDS-protein complex precipitation using potassium acetate, (3) gDNA isolation by two chloroform phase separations, (4) and gDNA capture/purification using a 1X Sera-Mag™ SpeedBead and 0.45X AMPure® PB double SPRI cleanup/size selection. Outcomes from 65–100 mg of fresh-frozen tissue homogenates are generally sufficient quantities (1-20 μg) of high purity, ultra HMW (uHMW; 100 kb+) gDNA adequate for multiple high-quality long-read sequencing events. However, outcome success is dependent on the plant species, tissue type and sample quality used. The output of this protocol is uHMW gDNA, which depending upon yield and genome size of the species can be directed downstream towards HMW DNA Pooling, HMW DNA Fragmentation: Diagenode Megaruptor®3 for LI PacBio or HMW DNA Fragmentation: g-Tube for ULI PacBio. Acronyms HMW: high molecular weight uHMW: ultra high molecular weight gDNA: genomic DNA SPRI: solid-phase reversible immobilisation LI: low input ULI: ultra-low input
Many children become HIV+ due to mother-to-child transmission, a risk that can be largely eliminated if infants ingest antiretroviral (ARV) medications immediately after birth. As most mothers in Africa deliver at home, the ARV must be provided at their last antenatal visit, sometimes months before birth. No current drug delivery system allows the mother to store the medication at home long enough to be effective. We propose a preserving, foilized, polyethylene pouch to be pre-dosed and sealed by a pharmacist for later delivery to the newborn. Pouches were filled with 0.6 ml of Nevirapine (NVP). Thirty-three pouches were immediately studied to measure the impact of medication handling (oxygen, light, filling and sealing the pouches). The remaining samples were stored for up to one year at three storage conditions (25°C/60%RH, 30°C/65%RH, and 40°C/75%RH). Every two months, moisture loss, preservative concentration, impurity concentrations and NVP concentration were measured. Flora and fauna challenges were conducted. The pouch nearly eliminated moisture loss: pouches lost less than 0.7% of their weight over twelve months. As expected, exposing the medication to light, oxygen, and handling significantly affected the sacrificial preservative concentrations (Propyl paraben dropped 38%, Methyl paraben dropped 12% at time point zero). However, after the initial time point, preservative levels were stable in the package over twelve months under all storage conditions (4.1% average concentration drop), leaving sufficient preservatives to protect the medication. The concentration of NVP changed an average of only 1.3% over all storage conditions and times points (maximum 1.4%). We conclude that the foilized polyethylene pouch can preserve NVP, and perhaps other ARV’s, for up to one year.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)