The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source-peripheral blood-has the potential to expedite the advances of ...iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34(+) cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3(-)/CD19(-) cells or T/B cell-depleted MNCs for 4-6 days led to the generation of 20-30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.
A summary of research on the impact of recreational catch-and-release angling for elasmobranchs, covering biochemical disruption- and physical injury-related impacts, and reporting associated ...mortality rates and sub-lethal consequences. Research findings are used to provide recommendations to policy makers and the angling community on how to enhance angling sustainability.
Abstract
Until relatively recently commercial fisheries have been considered the main driving factor for elasmobranch population declines. However, this belief has begun to shift with the realization that recreational elasmobranch catches may equal or exceed commercial catches in some regions. Many recreational angling fisheries for elasmobranchs involve high participation in catch-and-release angling practices. However, high release rates may not necessarily equate to high survival rates. Therefore, to assist accurate assessment of the potential impact of recreational angling on elasmobranchs, we attempted to summarize and integrate currently available information on specific risk factors associated with recreational angling, alongside associated mortality rates, as well as information on angler behaviour as it relates to identified risk factors. We categorized the major angling-related effects into two groups: injury-induced effects; and biochemical disruption-induced effects; providing a summary of each group and outlining the main lethal and sub-lethal outcomes stemming from these. These outcomes include immediate and delayed post-release mortality, behavioural recovery periods (which may in-turn confer increased predation risks), chronic health impacts and capture-induced parturition and abortion. Additionally, we detailed a range of angling practices and equipment, including hook-type, hook removal and emersion (i.e. air exposure), as well as inter- and intra-specific factors, including aerobic scope, respiratory mode, body size and species-specific behaviours, which are likely to influence injury and/or mortality rates and should therefore be considered when assessing angling-related impacts. We then utilized these data to provide a range of actionable recommendations for both anglers and policymakers which would serve to reduce the population-level impact of recreational angling on these enigmatic animals.
DNA methylation, a prototypical epigenetic modification implicated in gene silencing, occurs in many eukaryotes and plays a significant role in the etiology of diseases such as cancer. The ...filamentous fungus
places DNA methylation at regions of constitutive heterochromatin such as in centromeres and in other A:T-rich regions of the genome, but this modification is dispensable for normal growth and development. This and other features render
an excellent model to genetically dissect elements of the DNA methylation pathway. We implemented a forward genetic selection on a massive scale, utilizing two engineered antibiotic-resistance genes silenced by DNA methylation, to isolate mutants
efective
n
ethylation (
). Hundreds of potential mutants were characterized, yielding a rich collection of informative alleles of 11 genes important for DNA methylation, most of which were already reported. In parallel, we characterized the pairwise interactions in nuclei of the DCDC, the only histone H3 lysine 9 methyltransferase complex in Neurospora, including those between the DIM-5 catalytic subunit and other complex members. We also dissected the N- and C-termini of the key protein DIM-7, required for DIM-5 histone methyltransferase localization and activation. Lastly, we identified two alleles of a novel gene,
- a homolog of Clr5 in
- that is not essential for DNA methylation, but is necessary for repression of the antibiotic-resistance genes used in the selection, which suggests that both DIM-10 and DNA methylation promote silencing of constitutive heterochromatin.
•Stress Fractures in elderly patients are rarely discussed.•Vitamin D deficiency, osteoporosis, and weight are important comorbidities to consider for these stress fractures.•Have a high index of ...suspicion when multiple risk factors are observed in a patient.•Patients can be treated non-operatively or with minimally invasive procedures if the issue is identified early.
Early intervention in femoral neck stress fractures (FNSFs) can be self- limiting, but they have an insidious presentation. High index of suspicion for an occult fracture is necessary to avoid bilateral progression and/or operative interventions.
A 83-year-old female, non-athlete, presents with multiple comorbidities and progressive bilateral FNSFs. Initially the patient presented with groin pain and radiographs demonstrated a non-displaced compression type fracture of the right femoral neck without any inciting events. Subsequently, during a post-operative visit of her right hip, a work-up for new onset contralateral groin pain revealed a non-displaced tension type fracture on the left femoral neck. Both FNSFs were treated with percutaneous cannulated screw fixation.
Vitamin D deficiency, poor nutrition, and osteoporosis have been associated with developing stress fractures. This presents an interesting question of whether these frequently referenced risk factors play an interrelated role. Treatment algorithms are controversial, but have been successful in preventing the progression of occult stress fractures. Yet, identification of FNSFs represents a major challenge in diagnosis for clinicians.
This case report documents an uncommon fracture pattern in the elderly population. With an aging population, it is pertinent to avoid missed opportunities for prompt diagnosis and implementation of noninvasive methods of treatment. Therefore, paying attention to the risk factors with a high index of suspicion would be ideal.
Appropriate imaging modalities for screening, staging, and surveillance of patients with suspected and documented metastatic disease to bone include (99m)Tc bone scanning, MRI, CT, radiography, and ...2-(18)Ffluoro-2-deoxyglucose-PET. Clinical scenarios reviewed include asymptomatic stage 1 breast carcinoma, symptomatic stage 2 breast carcinoma, abnormal bone scan results with breast carcinoma, pathologic fracture with known metastatic breast carcinoma, asymptomatic well-differentiated and poorly differentiated prostate carcinoma, vertebral fracture with history of malignancy, non-small-cell lung carcinoma staging, symptomatic multiple myeloma, osteosarcoma staging and surveillance, and suspected bone metastasis in a pregnant patient. No single imaging modality is consistently best for the assessment of metastatic bone disease across all tumor types and clinical situations. In some cases, no imaging is indicated. The recommendations contained herein are the result of evidence-based consensus by the ACR Appropriateness Criteria((R)) Expert Panel on Musculoskeletal Radiology.
The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell ...replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34+ cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34+ cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.
Imaging of the diabetic foot is among the most challenging areas of radiology. The authors present a consensus of the suggested tests in several clinical scenarios, such as early neuropathy, ...soft-tissue swelling, skin ulcer, and suspected osteomyelitis. In most of these situations, magnetic resonance imaging (MRI) with or without contrast is the examination of choice. Most other imaging tests have complementary roles. For soft-tissue swelling or an ulcer, radiography and MRI with or without contrast are suggested. Bone scintigraphy with white blood cell scanning is used when MRI is contraindicated. In patients with diabetes without ulcers, radiography and MRI with or without contrast are suggested; bone scanning may be used when MRI is contraindicated.
The direct conversion of skin cells into somatic stem cells has opened new therapeutic possibilities in regenerative medicine. Here, we show that human induced mesenchymal stem cells （iMSCs） can be ...efficiently generated from cord blood （CB）- or adult peripheral blood （PB）-CD34cells by direct reprogramming with a single factor, OCT4. In the presence of a GSK3 inhibitor, 16% of the OCT4-transduced CD34 cells are converted into iMSCs within 2 weeks. Efficient direct reprogramming is achieved with both episomal vector-mediated transient OCT4 expression and lentiviral vector-mediated OCT4 transduction. The iMSCs express MSC markers, resemble bone marrow （BM）-MSCs in morphology, and possess in vitro multilineage differentiation capacity, yet have a greater proliferative capacity compared with BM-MSCs. Similar to BM-MSCs, the implanted iMSCs form bone and connective tissues, and are non-tumorigenic in mice. However, BM-MSCs do not, whereas iMSCs do form muscle fibers, indicating a potential functional advantage of iMSCs. In addition, we observed that a high level of OCT4 expression is required for the initial reprogramming and the optimal iMSC self-renewal, while a reduction of OCT4 expression is required for multilineage differentiation. Our method will contribute to the generation of patient-specific iMSCs, which could have applications in regenerative medicine. This discovery may also facilitate the development of strategies for direct conversion of blood cells into other types of cells of clinical importance.
Distribution of species across jurisdictional and physical boundaries poses a challenge to management and research, and these transboundary species tend to suffer more-severe population declines from ...fisheries exploitation. Large pelagic sharks like the porbeagle shark, Lamna nasus, are particularly vulnerable to anthropogenic pressures due to their life history characteristics and their highly migratory behaviour. However, limited knowledge of their precise spatio-temporal movements is particularly challenging for management in situations where jurisdictional boundaries change over small spatial scales. We used satellite tags to demonstrate that porbeagle sharks tagged in the northern Northeast (NE) Atlantic (n = 3), display inter-individual variation in behaviour. Tagged sharks undertook rapid horizontal movements (up to 100 km per day) while transiting through multiple physical habitats and management jurisdictions in a matter of days along different paths. The spatial scale of these movements is important now that the population is deemed in recovery and a new catch advice for porbeagle sharks has been issued by ICES for the first time since 2009 in the NE Atlantic. These movement data highlight the value of existing, and need for continued, regional collaboration to inform sustainable fisheries and conservation management. This is achieved by maximising research impact through cross border funding mechanisms to fill knowledge gaps of species’ life-history and ecology, and, in turn, improve respective outcomes for vulnerable and highly mobile shark species.
•Tagged porbeagle sharks in the Northeast Atlantic undertook rapid movements of up to 100 km per day.•These sharks crossed multiple jurisdictional, national, and physical boundaries over the course of the 4–12 months tracks.•Double tagging individuals using PSAT and SPOT tags helps better define vertical and horizontal space use of individuals.•Transboundary movements support the need for cross-border research to fill in knowledge gaps and inform management efforts.
Stargazer (
stg
) mutant mice fail to express stargazin transmembrane AMPA receptor regulatory protein γ2 (TARPγ2) and consequently experience absence seizure-like thalamocortical spike-wave ...discharges that pervade the hippocampal formation via the dentate gyrus (DG). As in other seizure models, the dentate granule cells of
stg
develop elaborate reentrant axon collaterals and transiently overexpress brain-derived neurotrophic factor. We investigated whether GABAergic parameters were affected by the
stg
mutation in this brain region. GABA
A
receptor (GABAR) α4 and β3 subunits were consistently upregulated, GABAR δ expression appeared to be variably reduced, whereas GABAR α1, β2, and γ2 subunits and the GABAR synaptic anchoring protein gephyrin were essentially unaffected. We established that the α4βγ2 subunit-containing, flunitrazepam-insensitive subtype of GABARs, not normally a significant GABAR in DG neurons, was strongly upregulated in
stg
DG, apparently arising at the expense of extrasynaptic α4βδ-containing receptors. This change was associated with a reduction in neurosteroid-sensitive GABAR-mediated tonic current. This switch in GABAR subtypes was not reciprocated in the tottering mouse model of absence epilepsy implicating a unique, intrinsic adaptation of GABAergic networks in
stg
.
Contrary to previous reports that suggested that TARPγ2 is expressed in the dentate, we find that TARPγ2 was neither detected in
stg
nor control DG. We report that TARPγ8 is the principal TARP isoform found in the DG and that its expression is compromised by the stargazer mutation. These effects on GABAergic parameters and TARPγ8 expression are likely to arise as a consequence of failed expression of TARPγ2 elsewhere in the brain, resulting in hyperexcitable inputs to the dentate.
PDF
