Feasibility assessment of inspiration breath-hold motion management for tumor tracking during cone-beam computed tomography for setup and radiotherapy in Veterinary Medicine: A pilot study.

Radiotherapy (RT) for lung or liver tumors can be challenging due to respiration-induced organ motion (RIOM). There are some methodological solutions to minimize RIOM. We explored a new approach to evaluate the feasibility and reproducibility of RIOM during RT with five total client-owned tumor-bearing animals using a remote-triggered breath-hold ventilator under general anesthesia during image acquisition and RT. There was one stereotactic body radiotherapy, one conventionally fractionated definitive intent, and three conventionally fractionated palliative intent RT cases. Based on repeated cone beam CT, there were no treatment table shifts required prior to initiating beam on. No clinically significant complications such as hypotension occurred during anesthesia. This technique appeared to be safe in this group of patients and was easily clinically implemented and highly reproducible. More complete follow-up data and larger studies are needed to evaluate clinical outcomes with this breath-hold ventilator technique in veterinary RT.

Contact Tracing and Exposure Investigation in Response to the First Case of Monkeypox Virus Infection in the United States During the 2022 Global Monkeypox Outbreak.

BACKGROUND: In May 2022, the first case of monkeypox virus (MPXV) infection in the United States in the current global outbreak was identified. As part of the public health and health care facility response, a contact tracing and exposure investigation was done. OBJECTIVE: To describe the contact tracing, exposure identification, risk stratification, administration of postexposure prophylaxis (PEP), and exposure period monitoring for contacts of the index patient, including evaluation of persons who developed symptoms possibly consistent with MPXV infection. DESIGN: Contact tracing and exposure investigation. SETTING: Multiple health care facilities and community settings in Massachusetts. PARTICIPANTS: Persons identified as contacts of the index patient. INTERVENTION: Contact notification, risk stratification, and symptom monitoring; PEP administration in a subset of contacts. MEASUREMENTS: Epidemiologic and clinical data collected through standard surveillance procedures at each facility and then aggregated and analyzed. RESULTS: There were 37 community and 129 health care contacts identified, with 4 at high risk, 49 at intermediate risk, and 113 at low or uncertain risk. Fifteen health care contacts developed symptoms during the monitoring period. Three met criteria for MPXV testing, with negative results. Two community contacts developed symptoms. Neither met criteria for MPXV testing, and neither showed disease progression consistent with monkeypox. Among 4 persons with high-risk exposures offered PEP, 3 elected to receive PEP. Among 10 HCP with intermediate-risk exposures for which PEP was offered as part of informed clinical decision making, 2 elected to receive PEP. No transmissions were identified at the conclusion of the 21-day monitoring period, despite the delay in recognition of monkeypox in the index patient. LIMITATION: Descriptions of exposures are subject to recall bias, which affects risk stratification. CONCLUSION: In a contact tracing investigation involving 166 community and health care contacts of a patient with monkeypox, no secondary cases were identified. PRIMARY FUNDING SOURCE: None.

Radiation Dose Audit Failures: Truth and Consequences.

The validation of a radiation sterilization dose involves an initial sterilization dose determination as well as maintenance of that sterilization dose. The procedures for maintenance of the sterilization dose typically include the periodic use of two types of tests: bioburden and dose audits. The details for the procedures are outlined in the ISO radiation sterilization standards. These documents also provide guidelines for recommended actions in response to the results of the two tests. The results for the dose audit are based on the number of positive tests of sterility (TOS) for products that have been irradiated at a verification or experimental dose. When the dose audit yields TOS positives, it is often thought that they indicate a sterilization failure and nonsterile product. The belief that any TOS positive is a failure is an incorrect assumption because of the statistical basis used for the determination of the sterilization dose. This article will outline the truth of what dose audit TOS positives mean in terms of the sterility assurance of product, as well as the consequences of TOS positives.

Impact of iodinated contrast shortage on contrast-associated acute kidney injury: a single center experience.

Background: In March 2022, a COVID-19 outbreak disrupted the global supply of iodine contrast media (ICM). Healthcare systems implemented contrast-saving strategies to maintain their remaining ICM supplies. This study sought to determine the impact of contrast shortage on the incidence of contrast-associated acute kidney injury (CA-AKI). Methods: This was a retrospective study of 265 patients undergoing 278 percutaneous coronary interventions (PCI) during 4-month periods prior to (9/1/2021 to 12/31/2021) and during (5/1/2022 to 8/31/2022) contrast shortage at a single center. The primary endpoint was the incidence of CA-AKI between study periods. Results: A total of 148 and 130 PCIs were performed before and during contrast shortage, respectively. The incidence of CA-AKI significantly decreased from 11.5% to 4.6% during contrast shortage (P = 0.04). During the shortage, average contrast volume per PCI was significantly lower (123 ± 62 mL vs 88 ± 46 mL, P < 0.001), while coronary imaging was significantly higher (34.3% vs 50%, P = 0.009) compared to preshortage. All-cause mortality at discharge was comparable between study periods (2.8% vs 3.3%, respectively; P = 0.90). Conclusion: The scarcity of ICM for PCI procedures in this single-center experience was associated with a significant increase in the utilization of intravascular imaging and a significant reduction in CA-AKI.

Evolution of a functionally intact but antigenically distinct DENV fusion loop.

A hallmark of dengue virus (DENV) pathogenesis is the potential for antibody-dependent enhancement, which is associated with deadly DENV secondary infection, complicates the identification of correlates of protection, and negatively impacts the safety and efficacy of DENV vaccines. Antibody-dependent enhancement is linked to antibodies targeting the fusion loop (FL) motif of the envelope protein, which is completely conserved in mosquito-borne flaviviruses and required for viral entry and fusion. In the current study, we utilized saturation mutagenesis and directed evolution to engineer a functional variant with a mutated FL (D2-FL), which is not neutralized by FL-targeting monoclonal antibodies. The FL mutations were combined with our previously evolved prM cleavage site to create a mature version of D2-FL (D2-FLM), which evades both prM- and FL-Abs but retains sensitivity to other type-specific and quaternary cross-reactive (CR) Abs. CR serum from heterotypic (DENV4)-infected non-human primates (NHP) showed lower neutralization titers against D2-FL and D2-FLM than isogenic wildtype DENV2 while similar neutralization titers were observed in serum from homotypic (DENV2)-infected NHP. We propose D2-FL and D2-FLM as valuable tools to delineate CR Ab subtypes in serum as well as an exciting platform for safer live-attenuated DENV vaccines suitable for naïve individuals and children.

Evolution of a Functionally Intact but Antigenically Distinct DENV Fusion Loop.

A hallmark of Dengue virus (DENV) pathogenesis is the potential for antibody-dependent enhancement, which is associated with deadly DENV secondary infection, complicates the identification of correlates of protection, and negatively impacts the safety and efficacy of DENV vaccines. ADE is linked to antibodies targeting the fusion loop (FL) motif of the envelope protein, which is completely conserved in mosquito-borne flaviviruses and required for viral entry and fusion. In the current study, we utilized saturation mutagenesis and directed evolution to engineer a functional variant with a mutated FL (D2-FL) which is not neutralized by FL-targeting monoclonal antibodies. The FL mutations were combined with our previously evolved prM cleavage site to create a mature version of D2-FL (D2-FLM), which evades both prM- and FL-Abs but retains sensitivity to other type-specific and quaternary cross-reactive (CR) Abs. CR serum from heterotypic (DENV4) infected non-human primates (NHP) showed lower neutralization titers against D2-FL and D2-FLM than isogenic wildtype DENV2 while similar neutralization titers were observed in serum from homotypic (DENV2) infected NHP. We propose D2-FL and D2-FLM as valuable tools to delineate CR Ab subtypes in serum as well as an exciting platform for safer live attenuated DENV vaccines suitable for naïve individuals and children.

Regulation of Parkinson's disease-associated genes by Pumilio proteins and microRNAs in SH-SY5Y neuronal cells.

Parkinson's disease is the second most common age-related, neurodegenerative disease. A small collection of genes has been linked to Parkinson's disease including LRRK2, SAT1, and SNCA, the latter of which encodes the protein alpha-synuclein that aggregates in Lewy bodies as a hallmark of the disease. Overexpression of even wild-type versions of these genes can lead to pathogenesis, yet the regulatory mechanisms that control protein production of the genes are not fully understood. Pumilio proteins belong to the highly conserved PUF family of eukaryotic RNA-binding proteins that post-transcriptionally regulate gene expression through binding conserved motifs in the 3' untranslated region (UTR) of mRNA targets known as PUF Recognition Elements (PREs). The 3'UTRs of LRRK2, SNCA and SAT1 each contain multiple putative PREs. Knockdown (KD) of the two human Pumilio homologs (Pumilio 1 and Pumilio 2) in a neurodegenerative model cell line, SH-SY5Y, resulted in increased SNCA and LRRK2 mRNA, as well as alpha-synuclein levels, suggesting these genes are normally repressed by the Pumilio proteins. Some studies have indicated a relationship between Pumilio and microRNA activities on the same target, especially when their binding sites are close together. LRRK2, SNCA, and SAT1 each contain several putative microRNA-binding sites within the 3'UTR, some of which reside near PREs. Small RNA-seq and microRNA qPCR assays were performed in both wild type and Pumilio KD SH-SY5Y cells to analyze global and differential microRNA expression. One thousand four hundred and four microRNAs were detected across wild type and Pumilio KD cells. Twenty-one microRNAs were differentially expressed between treatments, six of which were previously established to be altered in Parkinson's disease patient samples or research models. Expression of ten miRs predicted to target LRRK2 and SNCA was verified by RT-qPCR. Collectively, our results demonstrate that Pumilios and microRNAs play a multi-faceted role in regulating Parkinson's disease-associated genes.

Platelet-rich plasma for the treatment of coccydynia: a case report and review of regenerative medicine for coccydynia.

Aim: To describe the successful treatment of coccydynia using ultrasound-guided injection of platelet-rich plasma. Setting: Outpatient orthopedic practice. Patient: 17-year-old female with BMI of 42.6. Case description: The patient presented with 6 months of nontraumatic coccygeal pain exacerbated by sitting. Physical exam was significant for point-tenderness over the sacral hiatus and coccyx. A corticosteroid injection around the sacrococcygeal ligament was administered with immediate resolution of her pain following the injection with the anesthetic. The patient reported significant pain relief for 1 week. The superficial sacrococcygeal ligament was then treated with a platelet-rich plasma injection under US guidance. Results: The patient reported a 70% improvement in pain and sitting tolerance at 6 weeks. By 6 months post injection, her pain was 100% resolved, and she remained pain free at the 12-month follow-up. Conclusion: Platelet-rich plasma may be considered as a treatment option in patients with refractory coccydynia.

In vitro characterization of Pittsburgh compound-B binding to Lewy bodies.

Dementia with Lewy bodies (DLB) is pathologically characterized by the presence of alpha-synuclein-containing Lewy bodies within the neocortical, limbic, and paralimbic regions. Like Alzheimer's disease (AD), Abeta plaques are also present in most DLB cases. The contribution of Abeta to the development of DLB is unclear. [11C]-Pittsburgh compound B ([11C]-PIB) is a thioflavin-T derivative that has allowed in vivo Abeta burden to be quantified using positron emission tomography (PET). [11C]-PIB PET studies have shown similar high cortical [11C]-PIB binding in AD and DLB subjects. To establish the potential binding of PIB to alpha-synuclein in DLB patients, we characterized the in vitro binding of PIB to recombinant human alpha-synuclein and DLB brain homogenates. Analysis of the in vitro binding studies indicated that [3H]-PIB binds to alpha-synuclein fibrils but with lower affinity than that demonstrated/reported for Abeta(1-42) fibrils. Furthermore, [3H]-PIB was observed to bind to Abeta plaque-containing DLB brain homogenates but failed to bind to DLB homogenates that were Abeta plaque-free ("pure DLB"). Positive PIB fluorescence staining of DLB brain sections colocalized with immunoreactive Abeta plaques but failed to stain Lewy bodies. Moreover, image quantification analysis suggested that given the small size and low density of Lewy bodies within the brains of DLB subjects, any contribution of Lewy bodies to the [11C]-PIB PET signal would be negligible. These studies indicate that PIB retention observed within the cortical gray matter regions of DLB subjects in [11C]-PIB PET studies is largely attributable to PIB binding to Abeta plaques and not Lewy bodies.

Atherosclerotic Disease and its Relationship to Lumbar Degenerative Disk Disease, Facet Arthritis, and Stenosis With Computed Tomography Angiography.

BACKGROUND: The intervertebral disk is the largest avascular structure in the body. It relies on passive diffusion from arteries at the periphery of the disk for nutrition. Previous studies have suggested a correlation between vascular disease and lumbar degenerative disk disease (DDD), but the association with facet arthritis and stenosis has not been evaluated. OBJECTIVE: To evaluate the degree of lumbar artery stenosis, aortic atherosclerosis on computed tomography angiography, and its relationship to lumbar DDD, facet arthritis, and spinal canal stenosis. DESIGN: Retrospective case review. SETTING: Academic tertiary care hospital. PARTICIPANTS: Not applicable. METHODS: A total of 300 lumbar arteries (150 lumbar artery pairs of the first to fifth lumbar arteries) were evaluated on consecutive computed tomography angiography scans. Severity of vascular disease of lumbar arteries was documented as normal, mild, moderate, severe, or occluded. Aortic vascular disease was documented along the posterior wall where the lumbar arteries originate. MAIN OUTCOME MEASUREMENTS: The relationship between vascular disease with DDD, facet arthritis, and spinal canal stenosis was examined and further evaluated controlling for age. RESULTS: Lumbar artery and aortic atherosclerosis had a positive relationship with DDD, facet arthritis, and spinal stenosis that was statistically significant (P < .05) even after controlling for age. The correlation coefficient was greatest in the younger age group when looking at lumbar artery vascular disease with DDD (0.73, confidence interval 0.50-0.96, P < .0001) and aortic vascular disease with DDD (0.72, confidence interval 0.49-0.94, P < .0001). The correlation of vascular disease with facet arthritis and stenosis was not strong in the older age group. CONCLUSION: Atherosclerotic disease of the lumbar arteries and aorta correlated with lumbar DDD, facet arthritis, and spinal canal stenosis after we adjusted for age, although the correlation with facet arthritis and spinal canal stenosis was not as strong in the older age group. LEVEL OF EVIDENCE: IV.

Synthesizing Evidence in Public Policy Contexts: The Challenge of Synthesis When There Are Only a Few Studies.

For a variety of reasons, researchers and evidence-based clearinghouses synthesizing the results of multiple studies often have very few studies that are eligible for any given research question. This situation is less than optimal for meta-analysis as it is usually practiced, that is, by employing inverse variance weights, which allows more informative studies to contribute relatively more to the analysis. This article outlines the choices available for synthesis when there are few studies to synthesize. As background, we review the synthesis practices used in several projects done at the behest of governmental agencies and private foundations. We then discuss the strengths and limitations of different approaches to meta-analysis in a limited information environment. Using examples from the U.S. Department of Education's What Works Clearinghouse as case studies, we conclude with a discussion of Bayesian meta-analysis as a potential solution to the challenges encountered when attempting to draw inferences about the effectiveness of interventions from a small number of studies.

Early markers of retinal degeneration in rd/rd mice.

PURPOSE: In the rd/rd mouse, the cell death of rod photoreceptors has been correlated to abnormal levels of the cyclic nucleotide cGMP within photoreceptors. Given that cGMP is required for opening of the cationic channels, there is the possibility that a high cGMP concentration would maintain these channels open, at a high energy cost for the retina. METHODS: We investigated whether cation channels were maintained in an open state in the rd/rd mouse retina by determining the labeling pattern of an organic cationic probe (agmatine, AGB) which selectively enters cells through open cationic channels. The metabolic activity of the rd/rd mice was measured by assaying lactate dehydrogenase (LDH) activity in several tissues and Na+/K+ ATPase activity was measured as a function of development and degeneration of the retina. RESULTS: AGB neuronal labeling showed a systematic increase consistent with the known neuronal functional maturation in the normal retina. There was a significant higher AGB labeling of photoreceptors in the rd/rd mouse retina from P6 supporting the possibility of open cationic channels from an early age. There were no changes in the LDH activity of tissues that contain PDE6 or that have a similar LDH distribution as the retina. However, LDH activity was significantly higher in the rd/rd mouse retina than in those of control mice from birth to P6, and it dramatically decreased from P9 as the photoreceptors degenerated. The predominant LDH isoenzyme changes and loss after degeneration appeared to be LDH5. ATPase activity increased with age, reaching adult levels by P16. Unlike LDH activity, there was no significant difference in Na+/K+ ATPase activity between control and rd/rd mice at any age examined. CONCLUSIONS: We conclude that AGB is a useful marker of photoreceptors destined to degenerate. We discard the possibility of a generalized metabolic effect in the rd/rd mice. However, the elevated LDH activity present before photoreceptor differentiation indicated altered retinal metabolic activity that could not be associated with open cationic channels alone. Therefore, altered metabolic activity as indicated by LDH measurements in the retina appeared to be the earliest sensitive sign of future photoreceptor dysfunction in the rd/rd mice.

Contract sterilization and validation companies offer new options for sterile compounded preparations.

To ensure patient safety, adhere to good compounding practices, and safeguard against professional liability, the sterility of some compounded preparations must be assured. When in-house sterilization capabilities do not suffice, the intervention of a contract sterilization and/or validation company can assist in establishing the sterility, purity, and potency of the pharmaceuticals used in compounded preparations. In this article, three contract steriliziation companies and two contract validation firms are profiled, methods that compounders can use to best interface with those companies are presented, and sterilization and validation processes are explained and compared.

Utilization and cell-surface binding of hemin by Histoplasma capsulatum.

Histoplasma capsulatum, a dimorphic fungus capable of causing severe respiratory illness in immunocompromised individuals, resides in macrophages during mammalian infection. Previous studies suggest that siderophore-mediated iron transport may be important for the acquisition of iron from transferrin while the organism resides in macrophages. However, iron is also present as hemin in the intracellular environment of the macrophage and may serve as a major source of iron during infection. Thus the ability of H. capsulatum to use hemin and heme-containing compounds was examined. Histoplasma capsulatum G217B was iron-starved by adding the iron chelator deferoxamine mesylate to the culture. The addition of 10 microM hemin in the presence of deferoxamine mesylate restored growth to the levels seen in the absence of the chelator. Histoplasma capsulatum was also cultivated in an iron-limited, chemically defined medium without the addition of chelators and it was determined that the organism could also use hemoglobin as a sole source of iron. The method of iron internalization from heme was examined by measuring hemin binding to the yeast-cell surface. The ability of H. capsulatum to bind hemin was related to the nutritional status of the cells. Cells grown under iron-limited conditions bound more heme to the cell surface than did cells grown in medium without chelator. Pretreatment of iron-starved cells with proteinase K eliminated the ability of the organism to bind hemin. Additionally, the pre-incubation of iron-starved H. capsulatum with hemin eliminated the ability of these cells to remove hemin from the solution, although pre-incubation of cells with the iron-free form of hemin, protoporphyrin IX, only modestly affected the ability of the organism to bind hemin. These results suggest that H. capsulatum uses hemin as a sole source of iron and that one mechanism of iron acquisition involves a cell-surface receptor for hemin.

Localization of NMDA receptor subunits and mapping NMDA drive within the mammalian retina.

Glutamate is a major neurotransmitter in the retina and other parts of the central nervous system, exerting its influence through ionotropic and metabotropic receptors. One ionotropic receptor, the N-methyl-D-aspartate(NMDA) receptor, is central to neural shaping, but also plays a major role during neuronal development and in disease processes. We studied the distribution pattern of different subunits of the NMDA receptor within the rat retina including quantifying the pattern of labelling for all the NRI splice variants, the NR2A and NR2B subunits. The labelling pattern for the subunits was confined predominantly in the outer two-thirds of the inner plexiform layer. We also wanted to probe NMDA receptor function using an organic cation, agmatine (AGB); a marker for cation channel activity. Although there was an NMDA concentration-dependent increase in AGB labelling of amacrine cells and ganglion cells, we found no evidence of functional NMDA receptors on horizontal cells in the peripheral rabbit retina, nor in the visual streak where the type A horizontal cell was identified by GABA labelling. Basal AGB labelling within depolarizing bipolar cells was also noted. This basal bipolar cell AGB labelling was not modulated by NMDA and was completely abolished by the use of L-2-amino-4-phosphono-butyric acid,which is known to hyperpolarize retinal depolarizing bipolar cells. AGB is therefore not only useful as a probe of ligand-gated drive, but can also identify neurons that have constitutively open cationic channels. In combination,the NMDA receptor subunit distribution pattern and the AGB gating experiments strongly suggests that this ionotropic glutamate receptor is functional in the cone-driven pathway of the inner retina.

Localization and expression of the glutamate transporter, excitatory amino acid transporter 4, within astrocytes of the rat retina.

Mechanisms for the removal of glutamate are vital for maintaining normal function of the retina. Five excitatory amino acid transporters have been characterized to date from neuronal tissue, all of which are expressed within the retina except excitatory amino acid transporter 4 (EAAT4). In this study we examined the expression and localization of the glutamate transporter EAAT4 in the rat retina using RT-PCR and immunocytochemistry. RT-PCR using rat EAAT4 specific primers revealed a prominent 296-bp product in the retina, cortex and cerebellum. The identity of the EAAT4 fragment was confirmed by DNA sequencing. We examined the tissue expression levels of EAAT4 in cortex, retina and cerebellum using real-time PCR. The highest expression level was found in the cerebellum. Expression in the cortex was approximately 3.1% that of the cerebellum and the retina was found to have approximately 0.8% the total cerebellar EAAT4 content. In order to examine the specific cell types within the retina that express EAAT4, we performed immunocytochemistry using a rat EAAT4 specific antiserum. Cellular processes within the nerve fibre layer of the retina were intensely labelled for EAAT4. Double labelling EAAT4 with glial fibrillary acidic protein (GFAP) revealed extensive colocalization indicating that EAAT4 is localized within astrocytes within the retina. Double labelling of EAAT4 and the glutamate transporter EAAT1 (GLAST) revealed extensive colocalization suggesting that astrocytes in the retina express at least two types of glutamate transporters. These results suggest that astrocytes within the retina are well placed to provide mechanisms for glutamate removal as well as controlling cellular excitability.

Mapping photoreceptor and postreceptoral labelling patterns using a channel permeable probe (agmatine) during development in the normal and RCS rat retina.

The aim of this study was to determine whether agmatine, a channel permeable probe, can identify photoreceptor dysfunction in the Royal College of Surgeons (RCS) retina at an earlier stage to that shown by apoptosis or anatomical markers, and also characterize the neurochemical development of the inner retina in the normal and degenerating rat. We used isolated retinas at different ages incubated in physiological media containing agmatine. Subsequently, postembedding immunocytochemistry was used to determine the number of labelled photoreceptors and the labelling pattern within postreceptoral neurons. Agmatine labelling patterns revealed a sequential development of retinal neurons beginning at postnatal day (PND) 11/12 with most horizontal cells, a few ganglion and amacrine cells, showing a strong signal. The neurochemical development progressed rapidly, and reflects to a large part the known distribution of glutamate receptors, with inner nuclear labelling being evident by PND14, continuing with the same pattern of labelling in adulthood for the control retina. The RCS retina showed markedly reduced agmatine labelling in the inner retina at PND20. A rapid increase in photoreceptor AGB labelling was evident during the degeneration phase. Multiple samples at PND14 and PND16 confirmed a significant increase of labelled photoreceptors in the RCS retina.