Biodiversity patterns of the coral reef cryptobiota around the Arabian Peninsula.

The Arabian Peninsula accounts for approximately 6% of the world's coral reefs. Some thrive in extreme environments of temperature and salinity. Using 51 Autonomous Reef Monitoring Structure (ARMS), a standardized non-destructive monitoring device, we investigated the spatial patterns of coral reef cryptobenthic diversity in four ecoregions around the Arabian Peninsula and analyzed how geographical and/or environmental drivers shape those patterns. The mitochondrial cytochrome c oxidase subunit I (COI) gene was used to identify Amplicon Sequence Variants and assign taxonomy of the cryptobenthic organisms collected from the sessile and mobile fractions of each ARMS. Cryptobenthic communities sampled from the two ecoregions in the Red Sea showed to be more diverse than those inhabiting the Arabian (Persian) Gulf and the Gulf of Oman. Geographic distance revealed a stronger relationship with beta diversity in the Mantel partial correlation than environmental distance. However, the two mobile fractions (106-500 µm and 500-2000 µm) also had a significant correlation between environmental distance and beta diversity. In our study, dispersal limitations explained the beta diversity patterns in the selected reefs, supporting the neutral theory of ecology. Still, increasing differences in environmental variables (environmental filtering) also had an effect on the distribution patterns of assemblages inhabiting reefs within short geographic distances. The influence of geographical distance in the cryptofauna assemblages makes these relevant, yet usually ignored, communities in reef functioning vulnerable to large scale coastal development and should be considered in ecosystem management of such projects.

Hot Carrier Thermalization and Josephson Inductance Thermometry in a Graphene-Based Microwave Circuit.

Due to its exceptional electronic and thermal properties, graphene is a key material for bolometry, calorimetry, and photon detection. However, despite graphene's relatively simple electronic structure, the physical processes responsible for the heat transport from the electrons to the lattice are experimentally still elusive. Here, we measure the thermal response of low-disorder graphene encapsulated in hexagonal boron nitride by integrating it within a multiterminal superconducting microwave resonator. The device geometry allows us to simultaneously apply Joule heat power to the graphene flake while performing calibrated readout of the electron temperature. We probe the thermalization rates of both electrons and holes with high precision and observe a thermalization scaling exponent not consistent with cooling through the graphene bulk and argue that instead it can be attributed to processes at the graphene-aluminum interface. Our technique provides new insights into the thermalization pathways essential for the next-generation graphene thermal detectors.

Association of Brain Natriuretic Peptide Levels at Time of Injury with Morbidity and Mortality in Patients with Surgically Treated Hip Fractures.

An elevated brain natriuretic peptide (BNP) level has been shown to be associated with mortality and cardiac events in cardiac surgery, but its utility in the prediction of morbidity and mortality in hip fracture surgery is unknown. The primary aim of this study was to determine if there is a difference in BNP level at the time of injury between patients who do and do not develop complications after hip fracture surgery. The secondary aim was to determine if there is a predictive relationship between complications associated with the initial BNP level and mortality. Methods: A retrospective chart review of 455 hip fractures in patients ≥60 years old that were operatively treated between February 2014 and July 2018 was performed. Patients were included if they had a BNP level within 48 hours after injury (BNPi). Specific perioperative (≤7 days), 30-day, 1-year, and 2-year postoperative complications were recorded. Wilcoxon rank-sum tests were used to determine if higher BNPi values were associated with greater morbidity. The complications associated with higher BNPi values were further analyzed to assess if they were predictive of mortality, using univariate and multivariable analyses. Results: Higher BNPi was significantly associated with greater morbidity at all postoperative time points and with higher mortality at 1 and 2 years postoperatively. Furthermore, several complications including cardiac failure or exacerbation and altered mental status were associated with mortality at all time points in univariate analysis and at many time points in multivariable analysis. Conclusions: Patients with higher BNPi levels were more likely to develop complications up to 1 year postoperatively, and several of these complications were associated with increased mortality. Future studies to determine if delaying surgery until BNP levels are normalized or lowered may help guide management, and may be useful in determining the need for further medical optimization. Future studies aimed at defining a threshold BNP value at the time of injury may also help in better managing patients preoperatively. Level of Evidence: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Anxiety, depression, and sleep-related outcomes of glaucoma patients: systematic review and meta-analysis.

OBJECTIVE: This study aims to synthesize data quantifying the prevalence and severity of common psychological conditions in patients with glaucoma. DESIGN: Systematic review and meta-analysis. METHODS: Databases including Ovid MEDLINE, CINAHL, EMBASE, PsycINFO, Cochrane Library, Web of Science, Open Grey, and ProQuest Theses and dissertations were searched. Two reviewers independently assessed and screened all studies, followed by quality assessment of included studies using the modified Downs and Black checklist. Data were pooled using fixed-effect and random-effects models. RESULTS: Of 2067 studies identified by the search strategy, 57 passed full-text screening, and 45 studies (4 995 538 subjects) were eligible for analysis. Overall, the prevalence of depression (effect size [ES] = 0.19, 95% CI 0.16-0.23; n = 31), anxiety (ES = 0.25, 95% CI 0.21-0.30; n = 18), and sleep disorders (ES = 0.47, 95% CI 0.26-0.68; n = 7) were high in patients with glaucoma. Similarly, symptomatic measurements of depression (standardized mean difference [SMD] = 0.46, 95% CI 0.19-0.73), anxiety (SMD = 0.44, 95% CI 0.08-0.81), and sleep quality (SMD = 0.72, 95% CI 0.22-1.21) were significant in glaucoma patients. CONCLUSIONS: A higher prevalence and severity of depression, anxiety, and sleep disorders was experienced in patients with glaucoma compared with patients without glaucoma. Caregivers as well as health care providers may need to be aware of unique psychological and social stressors placed on glaucoma patients.

Neonatal Staphylococcus Aureus Sepsis: a 20-year Western Australian experience.

OBJECTIVES: The purpose of this study was to characterise neonatal Staphylococcus aureus (SA) sepsis in Western Australia (WA) between 2001 and 2020 at the sole tertiary neonatal intensive care unit (NICU), examine risk factors for sepsis in the cohort, and compare short- and long-term outcomes to control infants without any sepsis. METHODS: Retrospective cohort study at the Neonatal Directorate at King Edward Memorial Hospital (KEMH) and Perth Children's Hospital, using electronic databases and patient medical records. RESULTS: The overall incidence of SA sepsis was 0.10 per 1000 live births (62/614207). From 2001 to 2010 the incidence was 0.13/1000 live births, reducing to 0.07/1000 live births from 2011 to 2020. SA was most frequently isolated from endotracheal aspirates, and infants with SA sepsis had longer median duration of ventilatory support than those without any sepsis (31 days vs 18 days respectively, p < 0.001). In our cohort, SA sepsis was associated with worse neurodevelopmental outcomes compared to infants without any sepsis. CONCLUSIONS: The incidence of neonatal SA sepsis has reduced over the last 20 years, suggesting potential effectiveness of the preventative interventions implemented. Endotracheal tube (ETT) colonisation and prolonged ventilation may be under-recognised as potential sources of SA infection. Our study suggests SA sepsis may negatively impact neurodevelopmental outcomes.

Palmitoylethanolamide (PEA) Inhibits SARS-CoV-2 Entry by Interacting with S Protein and ACE-2 Receptor.

Lipids play a crucial role in the entry and egress of viruses, regardless of whether they are naked or enveloped. Recent evidence shows that lipid involvement in viral infection goes much further. During replication, many viruses rearrange internal lipid membranes to create niches where they replicate and assemble. Because of the close connection between lipids and inflammation, the derangement of lipid metabolism also results in the production of inflammatory stimuli. Due to its pivotal function in the viral life cycle, lipid metabolism has become an area of intense research to understand how viruses seize lipids and to design antiviral drugs targeting lipid pathways. Palmitoylethanolamide (PEA) is a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α) agonist that also counteracts SARS-CoV-2 entry and its replication. Our work highlights for the first time the antiviral potency of PEA against SARS-CoV-2, exerting its activity by two different mechanisms. First, its binding to the SARS-CoV-2 S protein causes a drop in viral infection of ~70%. We show that this activity is specific for SARS-CoV-2, as it does not prevent infection by VSV or HSV-2, other enveloped viruses that use different glycoproteins and entry receptors to mediate their entry. Second, we show that in infected Huh-7 cells, treatment with PEA dismantles lipid droplets, preventing the usage of these vesicular bodies by SARS-CoV-2 as a source of energy and protection against innate cellular defenses. This is not surprising since PEA activates PPAR-α, a transcription factor that, once activated, generates a cascade of events that leads to the disruption of fatty acid droplets, thereby bringing about lipid droplet degradation through ß-oxidation. In conclusion, the present work demonstrates a novel mechanism of action for PEA as a direct and indirect antiviral agent against SARS-CoV-2. This evidence reinforces the notion that treatment with this compound might significantly impact the course of COVID-19. Indeed, considering that the protective effects of PEA in COVID-19 are the current objectives of two clinical trials (NCT04619706 and NCT04568876) and given the relative lack of toxicity of PEA in humans, further preclinical and clinical tests will be needed to fully consider PEA as a promising adjuvant therapy in the current COVID-19 pandemic or against emerging RNA viruses that share the same route of replication as coronaviruses.

Clustering in a newly forming social network by subjective perceptions of loneliness.

Objectives: To determine whether first-year college students cluster in networks based on subjective perceptions of loneliness. Participants: 492 first-year Notre Dame students completed surveys across two semesters and provided communication data used to reconstruct their social networks. Methods: Subjective perceptions of loneliness are measured using the Social and Emotional Loneliness Scale for Adults (SELSA). Correlations between an individual's loneliness and the average loneliness of their alters are compared to associations in random networks created using a rewiring algorithm to determine statistical significance. Results: During their first semester, students are more likely than chance to form ties with other students with similar levels of family and romantic loneliness. In their second semester, students cluster on romantic loneliness but not on family or social loneliness. Conclusions: Students are more likely than chance to form ties with people with similar self-perceived levels of loneliness, but only for certain types of loneliness and during certain periods.

Isolation and Electrophysiology of Murine Sympathetic Postganglionic Neurons in the Thoracic Paravertebral Ganglia.

The thoracic paravertebral sympathetic chain postganglionic neurons (tSPNs) represent the predominant sympathetic control of vascular function in the trunk and upper extremities. tSPNs cluster to form ganglia linked by an interganglionic nerve and receive multisegmental convergent and divergent synaptic input from cholinergic sympathetic preganglionic neurons of the spinal cord (Blackman and Purves, 1969; Lichtman et al., 1980 ). Studies in the past have focused on cervical and lumbar chain ganglia in multiple species, but few have examined the thoracic chain ganglia, whose location and diminutive size make them less conducive to experimentation. Seminal studies on the integrative properties of preganglionic axonal projections onto tSPNs were performed in guinea pig (Blackman and Purves, 1969; Lichtman et al., 1980 ), but as mice have become the accepted mammalian genetic model organism, there is need to reproduce and expand on these studies in this smaller model. We describe an ex vivo approach that enables electrophysiological, calcium imaging, and optogenetic assessment of convergence, divergence, and studies on pre- to postganglionic synaptic transmission, as well as whole-cell recordings from individual tSPNs. Preganglionic axonal connections from intact ventral roots and interganglionic nerves across multiple segments can be stimulated to evoke compound action potential responses in individual thoracic ganglia as recorded with suction electrodes. Chemical block of synaptic transmission differentiates spiking of preganglionic axons from synaptically-recruited tSPNs. Further dissection, including removal of the sympathetic chain, enables whole-cell patch clamp recordings from individual tSPNs for characterization of cellular and synaptic properties.

Quality of referrals for glaucoma assessment: a cross-sectional survey of clinical data and outcomes.

PURPOSE: This study assessed the completeness of clinical information provided by ophthalmological and optometric referrals to glaucoma specialists consulting for open-angle glaucoma (OAG). METHODS: A retrospective, cross-sectional study of 72 internal referrals for evaluation of OAG in a multispecialty group practice was performed. The quality of the referral was assessed based on: (1) the completeness of the clinical triad of intraocular pressure measurement, visual field (VF), and cup-to-disk ratio for each eye; (2) the availability of the data necessary to calculate an ocular hypertension treatment study (OHTS) score; and (3) the presence of retinal nerve fiber layer (RNFL) imaging by mean of optical coherence tomography. RESULTS: The clinical triad was available in 57% of referrals, whereas an OHTS score was calculable in 24% of referrals (p < 0.001); RNLF imaging was available in 51% of referrals (p = 0.859). The completeness of clinical information was similar for ophthalmological and optometric referrals. From the date of referral to the time of the consultation, there was a significant increase in the availability of the clinical triad (57-65%; p = 0.013) and the OHTS score (24-5%; p = 0.004) but not for RNFL imaging (51-56%; p = 0.618). The most common missing clinical information was VF testing, which was absent in 42% of referrals. CONCLUSIONS: Key clinical data necessary for effective diagnosis and staging of OAG was lacking for many patients referred to glaucoma specialists.

Autologous Hematopoietic Stem Cell Transplantation for Chronic Inflammatory Demyelinating Polyradiculoneuropathy.

BACKGROUND: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) refractory to conventional therapy can lead to marked disability and represents a therapeutic challenge. OBJECTIVE: To report five cases of treatment-refractory disabling CIDP treated with autologous hematopoietic stem cell transplantation (AHSCT). METHODS: This was a retrospective cohort study from a tertiary care referral center for both neuromuscular disease and AHSCT. Patients with CIDP treated with AHSCT between 2008 and 2020 were included. All patients had major persistent and disabling neuropathic deficits despite combinations of intensive immunosuppressive therapy. The primary outcome measures were: Medical Research Council sum score, Overall Neuropathy Limitations Scale and requirement for ongoing CIDP immunotherapy after transplantation. We also analyzed safety outcomes by documenting all severe AHSCT-related complications. RESULTS: Five patients with refractory CIDP underwent AHSCT. Three were classified as manifesting a typical syndrome, two were classified as the multifocal Lewis Sumner variant. The mean age at time of CIDP diagnosis was 33.4 years (range 24-46 years), with a median delay of 46 months (range 21-135 months) between diagnosis and AHSCT. The median follow-up period was 41 months. All five patients were able to wean off CIDP-related immunotherapy. Marked improvements in Medical Research Council scale and overall Neuropathy Limitations Scale were noted in 4/5 patients. One patient with longstanding neurogenic atrophy showed no improvement in disability scales. There were no treatment-related deaths or critical illnesses. CONCLUSIONS: AHSCT can achieve marked sustained clinical improvement of refractory CIDP and may allow for weaning off long-term complex immunotherapies.

Antibiotic pollution promotes dominance by harmful cyanobacteria: A case study examining norfloxacin exposure in competition experiments.

Cyanobacterial harmful algal blooms (cyanoHABs) in freshwater lakes across the globe are often combined with other stressors. Pharmaceutical pollution, especially antibiotics in water bodies, poses a potential hazard in aquatic ecosystems. However, how antibiotics influence the risk of cyanoHABs remains unclear. Here, we investigated the effects of norfloxacin (NOR), one of the most widely used antibiotics globally, to a bloom-forming cyanobacterium (Microcystis aeruginosa) and a common green alga (Scenedesmus quadricauda), under both mono- and coculture conditions. Taxon-specific responses to NOR were evaluated in monoculture. In addition, the growth rate and change in ratio of cyanobacteria to green algae when cocultured with exposure to NOR were determined. In monocultures of Microcystis, exposure to low concentrations of NOR resulted in decreases in biomass, chlorophyll a and soluble protein content, while superoxide anion content and superoxide dismutase activity increased. However, NOR at high concentration only slightly affected Scenedesmus. During the co-culture trials of Microcystis and Scenedesmus, the 5 µg · L-1 NOR treatment increased the ratio of Microcystis to co-cultured Scenedesmus by 47.2%. Meanwhile, although Scenedesmus growth was enhanced by 4.2% under NOR treatment in monoculture, it was conversely inhibited by 63.4% and 38.2% when co-cultured with Microcystis with and without NOR, respectively. Our results indicate that antibiotic pollution has a potential risk to enhance the perniciousness of cyanoHABs by disturbing interspecific interaction between cyanobacteria and green algae. These results reinforce the need for scientists and managers to consider the influence of xenobiotics in shaping the outcome of interactions among multiple species in aquatic ecosystems.

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for CYP2C9 and HLA-B Genotypes and Phenytoin Dosing: 2020 Update.

Phenytoin is an antiepileptic drug with a narrow therapeutic index and large interpatient pharmacokinetic variability, partly due to genetic variation in CYP2C9. Furthermore, the variant allele HLA-B*15:02 is associated with an increased risk of Stevens-Johnson syndrome and toxic epidermal necrolysis in response to phenytoin treatment. We summarize evidence from the published literature supporting these associations and provide therapeutic recommendations for the use of phenytoin based on CYP2C9 and/or HLA-B genotypes (updates on cpicpgx.org).

An Initiative to Improve Follow-up of Patients with Glaucoma.

Purpose: This study describes the implementation of an electronic medical record (EMR)-based initiative aimed at reducing the number of patients with glaucoma-related diagnoses lost to follow-up (LTF) and reviews its short-term outcomes. Design: Retrospective, comparative case series. Participants: Patients with glaucoma-related diagnoses seen 1 year prior at the Lahey Medical Center and who had not returned within the 6-month period between January 1, 2020, and June 30, 2020, which spanned the outbreak of the Coronavirus Disease 2019 (COVID-19) pandemic in the United States. Methods: An EMR-based tool was designed to identify patients suspected of being LTF with glaucoma-related diagnoses. Providers were enlisted to review the EMR for each of these patients and re-engage them, as appropriate. One month later, the initiative was evaluated by means of a retrospective chart review. Binary logistic regression analysis was used to identify demographic, clinical, and sociomedical factors associated with being LTF. Main Outcome Measures: Patients who completed a telemedicine or in-person appointment, or had a future scheduled or ordered return appointment, were considered re-engaged. Results: Of the 3551 patients seen during the study period, 384 patients were identified as LTF (11%), with 60 identifying COVID-19 as the reason for canceling their visit (16%). Patients who lived farther from the eye clinic (P < 0.001) or who had a history of canceling or missing an appointment (P < 0.001) were more likely to be LTF. Patients with open-angle glaucoma (P = 0.042) or who had completed a visual field (P < 0.001) or ophthalmic imaging (P < 0.001) within the past year were less likely to be LTF. One month after the re-engagement initiative, 124 LTF patients (32%) had been re-engaged (40% through telemedicine), 238 patients (62%) had future scheduling orders in place, and 22 patients (6%) had no active plan for future follow-up. Conclusions: An EMR-based tool is an effective method for identifying patients at risk of being LTF and provides an opportunity for providers to recall and re-engage patients. Use of telemedicine to recontact LTF patients shows promise of improving the management of glaucoma, enhancing clinical productivity, and documenting treatment plans, thereby potentially reducing medicolegal liability.

Differential Sensitivity of Wild-Type and BRAF-Mutated Cells to Combined BRAF and Autophagy Inhibition

BRAF inhibitors are insufficient monotherapies for BRAF-mutated cancer; therefore, we investigated which inhibitory pathway would yield the most effective therapeutic approach when targeted in combination with BRAF inhibition. The oncogenic BRAF inhibitor, PLX4720, increased basal autophagic flux in BRAF-mutated cells compared to wild-type (WT) BRAF cells. Interestingly, early autophagy inhibition improved the effectiveness of PLX4720 regardless of BRAF mutation, whereas late autophagy inhibition did not. Although ATG5 knockout led to PLX4720 resistance in both WT and BRAF-mutated cells, the MEK inhibitor trametinib exhibited a synergistic effect on PLX4720 sensitivity in WT BRAF cells but not in BRAF-mutated cells. Conversely, the prolonged inhibition of endoplasmic reticulum (ER) stress reduced basal autophagy in BRAF-mutated cells, thereby increasing PLX4720 sensitivity. Taken together, our results suggest that the combined inhibition of ER stress and BRAF may simultaneously suppress both pro-survival ER stress and autophagy, and may therefore be suitable for treatment of BRAF-mutated tumors whose autophagy is increased by chronic ER stress. Similarly, for WT BRAF tumors, therapies targeting MEK signaling may be a more effective treatment strategy. Together, this study presents a rational combination treatment strategy to improve the efficacy of BRAF inhibitors depending on BRAF mutation status.

Differential Sensitivity of Wild-Type and BRAF-Mutated Cells to Combined BRAF and Autophagy Inhibition

BRAF inhibitors are insufficient monotherapies for BRAF-mutated cancer; therefore, we investigated which inhibitory pathway would yield the most effective therapeutic approach when targeted in combination with BRAF inhibition. The oncogenic BRAF inhibitor, PLX4720, increased basal autophagic flux in BRAF-mutated cells compared to wild-type (WT) BRAF cells. Interestingly, early autophagy inhibition improved the effectiveness of PLX4720 regardless of BRAF mutation, whereas late autophagy inhibition did not. Although ATG5 knockout led to PLX4720 resistance in both WT and BRAF-mutated cells, the MEK inhibitor trametinib exhibited a synergistic effect on PLX4720 sensitivity in WT BRAF cells but not in BRAF-mutated cells. Conversely, the prolonged inhibition of endoplasmic reticulum (ER) stress reduced basal autophagy in BRAF-mutated cells, thereby increasing PLX4720 sensitivity. Taken together, our results suggest that the combined inhibition of ER stress and BRAF may simultaneously suppress both pro-survival ER stress and autophagy, and may therefore be suitable for treatment of BRAF-mutated tumors whose autophagy is increased by chronic ER stress. Similarly, for WT BRAF tumors, therapies targeting MEK signaling may be a more effective treatment strategy. Together, this study presents a rational combination treatment strategy to improve the efficacy of BRAF inhibitors depending on BRAF mutation status.

Decreased miR-150 in obesity-associated type 2 diabetic mice increases intraocular inflammation and exacerbates retinal dysfunction.

INTRODUCTION: Diabetic retinopathy (DR) is the leading cause of blindness among the working population in the USA. Current therapies, including anti-vascular endothelial growth factor treatments, cannot completely reverse the visual defects induced by DR. MicroRNA-150 (miR-150) is a regulator that suppresses inflammation and pathological angiogenesis. In patients with diabetes, miR-150 is downregulated. As chronic inflammation is a major contributor to the pathogenesis of DR, whether diabetes-associated decrease of miR-150 is merely associated with the disease progression or decreased miR-150 causes retinal inflammation and pathological angiogenesis is still unknown. RESEARCH DESIGN AND METHODS: We used high-fat diet (HFD)-induced type 2 diabetes (T2D) in wild type (WT) and miR-150 knockout (miR-150-/-) mice for this study and compared retinal function and microvasculature morphology. RESULTS: We found that WT mice fed with an HFD for only 1 month had a significant decrease of miR-150 in the blood and retina, and retinal light sensitivity also decreased. The miR-150-/- mice on the HFD developed diabetes similar to that of the WT. At 7-8 months old, miR-150-/- mice under normal diet had increased degeneration of retinal capillaries compared with WT mice, indicating that miR-150 is important in maintaining the structural integrity of retinal microvasculature. Deletion of miR-150 worsened HFD-induced retinal dysfunction as early as 1 month after the diet regimen, and it exacerbated HFD-induced T2DR by further increasing retinal inflammation and microvascular degeneration. CONCLUSION: These data suggest that decreased miR-150 caused by obesity or diabetic insults is not merely correlated to the disease progression, but it contributes to the retinal dysfunction and inflammation, as well as the development of DR.

Prostate bed and organ-at-risk deformation: Prospective volumetric and dosimetric data from a phase II trial of stereotactic body radiotherapy after radical prostatectomy.

PURPOSE: Stereotactic body radiotherapy (SBRT) in the post-prostatectomy setting is investigational. A major concern is the deformable prostate bed clinical target volume (CTV) and the closely juxtaposed organs-at-risk (OARs). We report a volumetric and dosimetric analysis of kilovoltage cone-beam CT (CBCT) data from the first 18 patients enrolled on a phase II trial of post-prostatectomy SBRT. With instructions on bladder filling and rectal preparation, we hypothesized acceptable CTV coverage while minimal overdosing to OARs could be achieved. METHODS: All patients received 5 fractions of 6-6.8 Gy to the prostate bed. CBCT were taken prior to and halfway through each fraction. CTV and OARs were contoured for each CBCT. Changes in inter- and intra-fraction volume and dose were calculated. Relative changes in CTV V95%, bladder V32.5 Gy, and rectal V32.5 Gy and V27.5 Gy were evaluated. RESULTS: Interfraction CTV volume remained stable, with median change +5.69% (IQR -1.73% to +9.84%). CTV V95% exhibited median change -0.74% (IQR -9.15% to -0.07%). Volumetric and dosimetric changes were minor from interfraction rotation and intrafraction motion. CTV V95% was ≥93% in 13 of 18 (72%) patients; in the remaining five, median change was -14.09% (IQR -16.64% to -13.56%). Interfraction CTV volume change was significantly larger among patients with CTV V95% <93% (+25.04% vs. +2.85%, p = 0.002). CONCLUSIONS: With specific bladder and rectum filling protocols, CTV underdosing and overdosing to bladder and rectum are avoided in majority of patients. Changes in CTV shape may account for the underdosing that may be observed.

ATG5 knockout promotes paclitaxel sensitivity in drug-resistant cells via induction of necrotic cell death

Autophagy regulators are often effective as potential cancer therapeutic agents. Here, we investigated paclitaxel sensitivity in cells with knockout (KO) of ATG5 gene. The ATG5 KO in multidrug resistant v-Ha-ras -transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) was generated using the CRISPR/Cas9 technology. The qPCR and LC3 immunoblot confirmed knockout of the gene and protein of ATG5, respectively. The ATG5 KO restored the sensitivity of Ras-NIH 3T3/Mdr cells to paclitaxel. Interestingly, ATG5 overexpression restored autophagy function in ATG5 KO cells, but failed to rescue paclitaxel resistance. These results raise the possibility that low level of resistance to paclitaxel in ATG5 KO cells may be related to other roles of ATG5 independent of its function in autophagy. The ATG5 KO significantly induced a G2/M arrest in cell cycle progression. Additionally, ATG5 KO caused necrosis of a high proportion of cells after paclitaxel treatment. These data suggest that the difference in sensitivity to paclitaxel between ATG5 KO and their parental MDR cells may result from the disparity in the proportions of necrotic cells in both populations. Thus, our results demonstrate that the ATG5 KO in paclitaxel resistant cells leads to a marked G2/M arrest and sensitizes cells to paclitaxel-induced necrosis.

ATG5 knockout promotes paclitaxel sensitivity in drug-resistant cells via induction of necrotic cell death

Autophagy regulators are often effective as potential cancer therapeutic agents. Here, we investigated paclitaxel sensitivity in cells with knockout (KO) of ATG5 gene. The ATG5 KO in multidrug resistant v-Ha-ras -transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) was generated using the CRISPR/Cas9 technology. The qPCR and LC3 immunoblot confirmed knockout of the gene and protein of ATG5, respectively. The ATG5 KO restored the sensitivity of Ras-NIH 3T3/Mdr cells to paclitaxel. Interestingly, ATG5 overexpression restored autophagy function in ATG5 KO cells, but failed to rescue paclitaxel resistance. These results raise the possibility that low level of resistance to paclitaxel in ATG5 KO cells may be related to other roles of ATG5 independent of its function in autophagy. The ATG5 KO significantly induced a G2/M arrest in cell cycle progression. Additionally, ATG5 KO caused necrosis of a high proportion of cells after paclitaxel treatment. These data suggest that the difference in sensitivity to paclitaxel between ATG5 KO and their parental MDR cells may result from the disparity in the proportions of necrotic cells in both populations. Thus, our results demonstrate that the ATG5 KO in paclitaxel resistant cells leads to a marked G2/M arrest and sensitizes cells to paclitaxel-induced necrosis.

Dramatically Amplified Thoracic Sympathetic Postganglionic Excitability and Integrative Capacity Revealed with Whole-Cell Patch-Clamp Recordings.

Thoracic paravertebral sympathetic postganglionic neurons (tSPNs) comprise the final integrative output of the distributed sympathetic nervous system controlling vascular and thermoregulatory systems. Considered a non-integrating relay, what little is known of tSPN intrinsic excitability has been determined by sharp microelectrodes with presumed impalement injury. We thus undertook the first electrophysiological characterization of tSPN cellular properties using whole-cell recordings and coupled results with a conductance-based model to explore the principles governing their excitability in adult mice of both sexes. Recorded membrane resistance and time constant values were an order of magnitude greater than values previously obtained, leading to a demonstrable capacity for synaptic integration in driving recruitment. Variation in membrane resistivity was the primary determinant controlling cell excitability with vastly lower currents required for tSPN recruitment. Unlike previous microelectrode recordings in mouse which observed inability to sustain firing, all tSPNs were capable of repetitive firing. Computational modeling demonstrated that observed differences are explained by introduction of a microelectrode impalement injury conductance. Overall, tSPNs largely linearly encoded injected current magnitudes over a broad frequency range with distinct subpopulations differentiable based on repetitive firing signatures. Thus, whole-cell recordings reveal tSPNs have more dramatically amplified excitability than previously thought, with greater intrinsic capacity for synaptic integration and with the ability for maintained firing to support sustained actions on vasomotor tone and thermoregulatory function. Rather than acting as a relay, these studies support a more responsive role and possible intrinsic capacity for tSPNs to drive sympathetic autonomic function.