Prognostication in Prolonged and Chronic Disorders of Consciousness.

Patients with prolonged disorders of consciousness (DOCs) longer than 28 days may continue to make significant gains and achieve functional recovery. Occasionally, this recovery trajectory may extend past 3 (for nontraumatic etiologies) and 12 months (for traumatic etiologies) into the chronic period. Prognosis is influenced by several factors including state of DOC, etiology, and demographics. There are several testing modalities that may aid prognostication under active investigation including electroencephalography, functional and anatomic magnetic resonance imaging, and event-related potentials. At this time, only one treatment (amantadine) has been routinely recommended to improve functional recovery in prolonged DOC. Given that some patients with prolonged or chronic DOC have the potential to recover both consciousness and functional status, it is important for neurologists experienced in prognostication to remain involved in their care.

Association Between Ischemic Stroke Subtype and Stroke Severity: The Atherosclerosis Risk in Communities Study.

BACKGROUND AND OBJECTIVES: Disability after stroke occurs across ischemic stroke subtypes, with a suggestion that embolic strokes are more devastating. Whether this difference is as a result of differences in comorbidities or differences in severity at the time of the stroke event is not known. The primary hypothesis was that participants with embolic stroke would have more severe stroke at the time of admission and a higher risk of mortality, compared with thrombotic stroke participants even with consideration of confounders over time, with a secondary hypothesis that this association would differ by race and sex. METHODS: Atherosclerosis Risk in Communities (ARIC) study participants with incident adjudicated ischemic stroke, stroke severity and mortality data, and complete covariates were included. Multinomial logistic regression models determined the association between stroke subtype (embolic vs thrombotic) and admission NIH Stroke Scale (NIHSS) category (minor [≤5], mild [6-10], moderate [11-15], severe [16-20], and very severe [>20]) adjusted for covariates from visits most proximal to the stroke. Separate ordinal logistic models evaluated for interaction by race and sex. Adjusted Cox proportional hazard models estimated the association between stroke subtype and all-cause mortality (through December 31, 2019). RESULTS: Participants (N = 940) were mean age 71 years (SD = 9) at incident stroke, 51% female, and 38% Black. Using adjusted multinomial logistic regression, the risk of having a more severe stroke (reference NIHSS ≤5) was higher among embolic stroke vs thrombotic stroke patients, with a step-wise increase for embolic stroke patients when moving from mild (odds ratio [OR] 1.95, 95% CI 1.14-3.35) to very severe strokes (OR 4.95, 95% CI 2.34-10.48). After adjusting for atrial fibrillation, there was still a higher risk of having a worse NIHSS among embolic vs thrombotic strokes but with attenuation of effect (very severe stroke OR 3.91, 95% CI 1.76-8.67). Sex modified the association between stroke subtype and severity (embolic vs thrombotic stroke, p interaction = 0.03, per severity category, females OR 2.38, 95% CI 1.55-3.66; males OR 1.75, 95% CI 1.09-2.82). The risk of death (median follow-up 5 years, interquartile range 1-12) was also increased for embolic vs thrombotic stroke patients (hazard ratio 1.66, 95% CI 1.41-1.97). DISCUSSION: Embolic stroke was associated with greater stroke severity at the time of the event and a higher risk of death vs thrombotic stroke, even after careful adjustment for patient-level differences.

Safe Use of Cenobamate in Super Refractory Status Epilepticus: A Case Series.

Cenobamate is an effective new adjunctive antiseizure medication (ASM) for treatment resistant focal epilepsy. It has broad spectrum anticonvulsant activity and may be a useful medication for super refractory status epilepticus (SRSE), but has not yet been studied in generalized seizures or an inpatient setting. Here we describe 2 SRSE cases where cenobamate was added safely to other treatments. It was uptitrated slowly to reduce the risk of hypersensitivity reactions which have been observed previously with rapid increasing dosages. Both patients achieved seizure control and liberation from intensive care. They have remained seizure free with continued treatment and have not experienced any side effects attributable to cenobamate. Cenobamate warrants further examination in patients with refractory status epilepticus.

Surgical Management in Herpes Simplex Encephalitis: Illustrative Case Report and Systematic Review of the Literature.

BACKGROUND: Herpes simplex virus (HSV) is a common cause of viral encephalitis and can result in refractory seizures. Although HSV encephalitis (HSVE) is treated primarily with acyclovir, surgery can play a role in medically intractable cases. OBJECTIVE: To systematically review cases describing surgery for the treatment of severe HSVE. We also present an illustrative case of anterior temporal lobectomy (ATL) for refractory status epilepticus in a patient with unilateral HSVE. This case demonstrates one clinical context in which surgery can be a useful adjunct. METHODS: We performed a systematic review using PubMed and Google Scholar, including case reports and series describing surgical interventions for HSVE. Clinical data were extracted from 54 publications that incorporated 67 patient cases. RESULTS: Surgical decompression occurred at a wide range of times after the onset of illness, although most patients were operated on 4 or more days after HSVE symptoms began. Numerous reports indicated that decompressive craniectomy, temporal lobectomy, and hematoma removal could treat intractably elevated intracranial pressure because of HSVE with favorable long-term outcomes. We describe an additional case in which a 52-year-old woman with HSVE developed refractory right temporal lobe seizures. After ATL, the seizures resolved with significant clinical improvement. CONCLUSION: Surgical treatment can be a useful adjunct for treatment of HSVE. There is substantial variability in the timing of surgical decompression in patients with HSVE, which can be necessary up to approximately 3 weeks after illness onset. ATL should be considered for refractory status epilepticus in HSVE with a unilateral seizure focus.

Early withdrawal of life sustaining therapy in extracorporeal cardiopulmonary resuscitation (ECPR): Results from the Extracorporeal Life Support Organization registry.

AIMS: Although extracorporeal cardiopulmonary resuscitation (ECPR) improves survival outcomes in refractory cardiac arrest, morbidity and mortality remain significantly high. Information on causes of death in ECPR is limited; however, some evidence suggests withdrawal of life sustaining therapy (WLST) is a major factor in ECPR-associated mortality. We sought to describe the patients experiencing WLST after ECPR. METHODS: The international Extracorporeal Life Support Organization (ELSO) Registry was retrospectively queried for patients more than 18 years old supported with ECPR who underwent WLST due to family request from 2007 to 2017. These patients were split into groups for descriptive and multivariable analysis: early (WLST < 72 hours from cannulation) and routine WLST. RESULTS: Overall, 411 ECPR patients experienced WLST (median age 42 years IQR = 28-51; 31.7% female) over the 10-year period. 55.5% (n = 228) underwent early WLST with a median ECPR duration of 24 hours (IQR = 7-48) versus routine WLST (median = 147 hours; IQR = 105-238). In multivariable regression analysis, lower arterial blood gas pH (aOR = -3.1; 95% CI = 2.18-2.8; p = 0.04), arterial oxygen saturation (aOR = 1.12; 95% CI = 1.01-1.23; p = 0.02), and higher peak inspiratory pressure (aOR = 0.84; 95% CI = 0.71-1.00; p = 0.05) were independently associated with early WLST. Early WLST patients experienced higher rates of all ECMO-related complications except for infections. CONCLUSIONS: More than half of ECPR patients experienced early WLST within 72 hours. The patients with early WLST had worse markers of severe critical illness at 24 hours and experienced higher rates of complications. Further research should include an appropriate control group to better adjust confounders for ECPR-associated death and focus on prognostication.

Methods for stroke severity assessment by chart review in the Atherosclerosis Risk in Communities study.

Stroke severity is the most important predictor of post-stroke outcome. Most longitudinal cohort studies do not include direct and validated measures of stroke severity, yet these indicators may provide valuable information about post-stroke outcomes, as well as risk factor associations. In the Atherosclerosis Risk in Communities (ARIC) study, stroke severity data were retrospectively collected, and this paper outlines the procedures used and shares them as a model for assessment of stroke severity in other large epidemiologic studies. Trained physician abstractors, who were blinded to other clinical events, reviewed hospital charts of all definite/probable stroke events occurring in ARIC. In this analysis we included 1,198 ischemic stroke events occurring from ARIC baseline (1987-1989) through December 31, 2009. Stroke severity was categorized according to the National Institutes of Health Stroke Scale (NIHSS) score and classified into 5 levels: NIHSS ≤ 5 (minor), NIHSS 6-10 (mild), NIHSS 11-15 (moderate), NIHSS 16-20 (severe), and NIHSS > 20 (very severe). We assessed interrater reliability in a subgroup of 180 stroke events, reviewed independently by the lead abstraction physician and one of the four secondary physician abstractors. Interrater correlation coefficients for continuous NIHSS score as well as percentage of absolute agreement and Cohen Kappa Statistic for NIHSS categories were presented. Determination of stroke severity by the NIHSS, based on data abstracted from hospital charts, was possible for 97% of all ischemic stroke events. Median (25%-75%) NIHSS score was 5 (2-8). The distribution of NIHSS category was NIHSS ≤ 5 = 58.3%, NIHSS 6-10 = 24.5%, NIHSS 11-15 = 8.9%, NIHSS 16-20 = 4.7%, NIHSS > 20 = 3.6%. Overall agreement in the classification of severity by NIHSS category was present in 145/180 events (80.56%). Cohen's simple Kappa statistic (95% CI) was 0.64 (0.55-0.74) and weighted Kappa was 0.79 (0.72-0.86). Mean (SD) NIHSS score was 5.84 (5.88), with a median score of 4 and range 0-31 for the lead reviewer (rater 1) and mean (SD) 6.16 (6.10), median 4.5 and range 0-36 in the second independent assessment (rater 2). There was a very high correlation between the scores reported in both assessments (Pearson r = 0.90). Based on our findings, we conclude that hospital chart-based retrospective assessment of stroke severity using the NIHSS is feasible and reliable.

Population Characteristics and Markers for Withdrawal of Life-Sustaining Therapy in Patients on Extracorporeal Membrane Oxygenation.

OBJECTIVE: As survival with extracorporeal membrane oxygenation (ECMO) therapy improves, it is important to study patients who do not survive secondary to withdrawal of life-sustaining therapy (WLST). The purpose of the present study was to determine the population and clinical characteristics of those who experienced short latency to WLST. DESIGN: Retrospective cohort study. SETTING: Single academic hospital center. PARTICIPANTS: Adult ECMO patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: During the study period, 150 patients (mean age 54.8 ± 15.9 y, 43.3% female) underwent ECMO (80% venoarterial ECMO and 20% venovenous ECMO). Seventy-three (48.7%) had WLST from ECMO support (median five days), and 33 of those (45.2%) had early WLST (≤five days). Patients who underwent WLST were older (60.3 ± 15.3 y v 49.6 ± 14.7 y; p < 0.001) than those who did not undergo WLST and had greater body mass index (31.7 ± 7.6 kg/m2v 28.3 ± 5.5 kg/m2; p = 0.002), longer ECMO duration (six v four days; p = 0.01), and higher Acute Physiology and Chronic Health Evaluation (25 v 21; p < 0.001) and Sequential Organ Failure Assessment (12 v 11; p = 0.037) scores. Family request frequently (91.7%) was cited as part of the WLST decision. WLST patients experienced more chaplaincy (89% v 65%; p < 0.001), palliative care consults (53.4% v 29.9%; p = 0.003), and code status change (do not resuscitate: 83.6% v 7.8%; p < 0.001). CONCLUSIONS: Nearly 50% of ECMO patients underwent WLST, with approximately 25% occurring in the first 72 hours. These patients were older, sicker, and experienced a different clinical context. Unlike with other critical illnesses, neurologic injury was not a primary reason for WLST in ECMO patients.

Sustaining Gains in Diabetic Eye Screening: Outcomes from a Stakeholder-Based Implementation Program for Teleophthalmology in Primary Care.

Background: Teleophthalmology is a validated method for diabetic eye screening that is underutilized in U.S. primary care clinics. Even when made available to patients, its long-term effectiveness for increasing screening rates is often limited. Introduction: We hypothesized that a stakeholder-based implementation program could increase teleophthalmology use and sustain improvements in diabetic eye screening. Materials and Methods:We used the NIATx Model to test a stakeholder-based teleophthalmology implementation program, I-SITE at one primary care clinic (Main) and compared teleophthalmology use and diabetic eye screening rates with those of other primary care clinics (Outreach) within a U.S. multipayer health system where teleophthalmology was underutilized.Results:Teleophthalmology use increased post-I-SITE implementation (odds ratio [OR] = 5.73 [p < 0.001]), and was greater at the Main than at the Outreach clinics (OR = 10.0 vs. 1.69, p < 0.001). Overall diabetic eye screening rates maintained an increase from 47.4% at baseline to 60.2% and 64.1% at 1 and 2 years post-I-SITE implementation, respectively (p < 0.001). Patients who were younger (OR = 0.98 per year of age, p = 0.02) and men (OR = 1.98, p = 0.002) were more likely to use teleophthalmology than in-person dilated eye examinations for diabetic eye screening.Discussion: Our stakeholder-based implementation program achieved a significant increase in overall teleophthalmology use and maintained increased post-teleophthalmology diabetic eye screening rates. Conclusion: Stakeholder-based implementation may increase the long-term reach and effectiveness of teleophthalmology to reduce vision loss from diabetes. Our approach may improve integration of telehealth interventions into primary care.

High-dose hydroxocobalamin achieves biochemical correction and improvement of neuropsychiatric deficits in adults with late onset cobalamin C deficiency.

Cobalamin C (cblC) deficiency is the most common inborn error of intracellular cobalamin metabolism caused by pathogenic variant(s) in MMACHC and manifests with methylmalonic acidemia, hyperhomocysteinemia, and hypomethioninemia with a variable age of presentation. Individuals with late-onset cblC may be asymptomatic until manifesting neuropsychiatric symptoms, thromboembolic events, and renal disease. Although hydroxocobalamin provides a foundation for therapy, optimal dose regimen for adult patients has not been systematically evaluated. We report three adult siblings with late-onset cblC disease, and their biochemical and clinical responses to high-dose hydroxocobalamin. The 28-year-old proband presented with severe psychosis, progressive neurological deterioration, and deep venous thrombosis complicated by a pulmonary embolism. MRI studies identified lesions in the spinal cord, periventricular white matter, and basal ganglia. Serum homocysteine and methylmalonic acid levels were markedly elevated. Hydroxocobalamin at standard dose (1 mg/day) initially resulted in partial metabolic correction. A regimen of high-dose hydroxocobalamin (25 mg/day) together with betaine and folic acid resulted in rapid and sustainable biochemical correction, resolution of psychosis, improvement of neurological functions, and amelioration of brain and spinal cord lesions. Two siblings who did not manifest neuropsychiatric symptoms or thromboembolism achieved a satisfactory metabolic control with the same high-dose regimen. Hydroxocobalamin injection was then spaced out to 25 mg weekly with good and sustainable metabolic control. All three patients are compound heterozygotes for c.271dupA p.Arg91LysfsX14 and c.389A > G p.Tyr130Cys. This study highlights the importance of evaluating intracellular cobalamin metabolism in adults with neuropsychiatric manifestations and/or thromboembolic events, and demonstrates that high-dose hydroxocobalamin achieves rapid and sustainable metabolic control and improvement in neuropsychiatric outcomes in adults with late-onset cblC disease.

Identification of barriers, facilitators and system-based implementation strategies to increase teleophthalmology use for diabetic eye screening in a rural US primary care clinic: a qualitative study.

OBJECTIVE: Teleophthalmology for diabetic eye screening is an evidence-based intervention substantially underused in US multipayer primary care clinics, even when equipment and trained personnel are readily available. We sought to identify patient and primary care provider (PCP) barriers, facilitators, as well as strategies to increase teleophthalmology use. DESIGN: We conducted standardised open-ended, individual interviews and analysed the transcripts using both inductive and directed content analysis to identify barriers and facilitators to teleophthalmology use. The Chronic Care Model was used as a framework for the development of the interview guide and for categorising implementation strategies to increase teleophthalmology use. SETTING: A rural, US multipayer primary care clinic with an established teleophthalmology programme for diabetic eye screening. PARTICIPANTS: We conducted interviews with 29 participants (20 patients with diabetes and 9 PCPs). RESULTS: Major patient barriers to teleophthalmology use included being unfamiliar with teleophthalmology, misconceptions about diabetic eye screening and logistical challenges. Major patient facilitators included a recommendation from the patient's PCP and factors related to convenience. Major PCP barriers to referring patients for teleophthalmology included difficulty identifying when patients are due for diabetic eye screening and being unfamiliar with teleophthalmology. Major PCP facilitators included the ease of the referral process and the communication of screening results. Based on our results, we developed a model that maps where these key patient and PCP barriers occur in the teleophthalmology referral process. Patients and PCPs also identified implementation strategies to directly address barriers and facilitators to teleophthalmology use. CONCLUSIONS: Patients and PCPs have limited familiarity with teleophthalmology for diabetic eye screening. PCPs were expected to initiate teleophthalmology referrals, but reported significant difficulty identifying when patients are due for diabetic eye screening. System-based implementation strategies primarily targeting PCP barriers in conjunction with improved patient and provider education may increase teleophthalmology use in rural, US multipayer primary care clinics.

Factors influencing patient adherence with diabetic eye screening in rural communities: A qualitative study.

OBJECTIVE: Diabetic retinopathy remains the leading cause of blindness among working-age U.S. adults largely due to low screening rates. Rural populations face particularly greater challenges to screening because they are older, poorer, less insured, and less likely to receive guideline-concordant care than those in urban areas. Current patient education efforts may not fully address multiple barriers to screening faced by rural patients. We sought to characterize contextual factors affecting rural patient adherence with diabetic eye screening guidelines. RESEARCH DESIGN AND METHODS: We conducted semi-structured interviews with 29 participants (20 adult patients with type 2 diabetes and 9 primary care providers) in a rural, multi-payer health system. Both inductive and directed content analysis were performed. RESULTS: Factors influencing rural patient adherence with diabetic eye screening were categorized as environmental, social, and individual using the Ecological Model of Health. Major themes included limited access to and infrequent use of healthcare, long travel distances to obtain care, poverty and financial tradeoffs, trusting relationships with healthcare providers, family members' struggles with diabetes, anxiety about diabetes complications, and the burden of diabetes management. CONCLUSIONS: Significant barriers exist for rural patients that affect their ability to adhere with yearly diabetic eye screening. Many studies emphasize patient education to increase adherence, but current patient education strategies fail to address major environmental, social, and individual barriers. Addressing these factors, leveraging patient trust in their healthcare providers, and strategies targeted specifically to environmental barriers such as long travel distances (e.g. teleophthalmology) may fill crucial gaps in diabetic eye screening in rural communities.

Sodium bicarbonate cotransporter NBCe2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells.

RATIONALE: Salt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure. OBJECTIVE: To characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5. METHODS AND RESULTS: The expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte nuclear factor type 4A (HNF4A) to DNA was increased in hRPTCs carrying HV SLC4A5 rs7571842 but not rs10177833. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was abolished by HNF4A antagonists. CONCLUSION: NBCe2 activity is stimulated by an increase in intracellular sodium and is hyper-responsive in hRPTCs carrying HV SLC4A5 rs7571842 through an aberrant HNF4A-mediated mechanism.

Rituximab for subcutaneous delivery: Clinical management principles from a nursing perspective.

Nurses play an integral role in administering treatments to patients with non-Hodgkin's lymphomas. Intravenous (IV) rituximab was approved by the Australian Therapeutic Goods Administration in 1998, and a novel subcutaneous (SC) formulation was approved in 2014. Fixed-dose SC rituximab is highly concentrated; co-formulation with a fully human recombinant vorhyaluronidase alfa enzyme helps overcome the physiological barriers of the SC space, facilitating drug dispersion. Despite a different pharmacokinetic profile to the IV preparation, SC rituximab demonstrates a comparable efficacy/safety profile. Most frequently occurring rituximab-related adverse events include neutropenia, nausea and constipation, and administration-related reactions are more frequent with the SC preparation. Compared with IV, SC delivery reduces treatment times and nurse workload, and patients report greater comfort and convenience. This article sets out nursing considerations for optimal administration of SC rituximab, including premedication, drug handling/preparation, injection technique, after-care and management of adverse events, particularly administration-related reactions.

The sodium-bicarbonate cotransporter NBCe2 (slc4a5) expressed in human renal proximal tubules shows increased apical expression under high-salt conditions.

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake.

Fast, Ratiometric FRET from Quantum Dot Conjugated Stabilized Single Chain Variable Fragments for Quantitative Botulinum Neurotoxin Sensing.

Botulinum neurotoxin (BoNT) presents a significant hazard under numerous realistic scenarios. The standard detection scheme for this fast-acting toxin is a lab-based mouse lethality assay that is sensitive and specific, but slow (∼2 days) and requires expert administration. As such, numerous efforts have aimed to decrease analysis time and reduce complexity. Here, we describe a sensitive ratiometric fluorescence resonance energy transfer scheme that utilizes highly photostable semiconductor quantum dot (QD) energy donors and chromophore conjugation to compact, single chain variable antibody fragments (scFvs) to yield a fast, fieldable sensor for BoNT with a 20-40 pM detection limit, toxin quantification, adjustable dynamic range, sensitivity in the presence of interferents, and sensing times as fast as 5 min. Through a combination of mutations, we achieve stabilized scFv denaturation temperatures of more than 60 °C, which bolsters fieldability. We also describe adaptation of the assay into a microarray format that offers persistent monitoring, reuse, and multiplexing.

Exosomal transfer from human renal proximal tubule cells to distal tubule and collecting duct cells.

OBJECTIVES: Exosomes are 50-90nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have reported that human urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. This study examines exosomal transfer between cultured human renal proximal tubule cells (RPTCs) and from RPTCs to human distal tubule and collecting duct cells. DESIGN AND METHODS: For RPTC-to-RPTC exosomal transfer, we utilized 5 RPTC lines producing exosomes that were fluorescently labeled with exosomal-specific markers CD63-EGFP or CD9-RFP. Transfer between RPTCs was demonstrated by co-culturing CD63-EGFP and CD9-RFP stable clones and performing live confocal microscopy. For RPTC-to-distal segment exosomal transfer, we utilized 5 distal tubule and 3 collecting duct immortalized cell lines. RESULTS: Time-lapse videos revealed unique proximal tubule cellular uptake patterns for exosomes and eventual accumulation into the multivesicular body. Using culture supernatant containing exosomes from 3 CD9-RFP and 2 CD63-EGFP RPTC cell lines, all 5 distal tubule cell lines and all 3 collecting duct cell lines showed exosomal uptake as measured by microplate fluorometry. Furthermore, we found that RPTCs stimulated with fenoldopam (dopamine receptor agonist) had increased production of exosomes, which upon transfer to distal tubule and collecting duct cells, reduced the basal reactive oxygen species (ROS) production rates in those recipient cells. CONCLUSION: Due to the complex diversity of exosomal contents, this proximal-to-distal vesicular inter-nephron transfer may represent a previously unrecognized trans-renal communication system.

Prevention of alcohol-heightened aggression by CRF-R1 antagonists in mice: critical role for DRN-PFC serotonin pathway.

Alcohol can escalate aggressive behavior in a significant subgroup of rodents, humans, and nonhuman primates. The present study investigated whether blockade of corticotropin-releasing factor receptor type 1 (CRF-R1) could prevent the emergence of alcohol-heightened aggression in mice. The serotonin (5-HT) pathway from the dorsal raphe nucleus (DRN) to the medial prefrontal cortex (mPFC) by CRF-R1 was investigated as a possible target for the prevention of alcohol-heightened aggressive behavior. Male CFW mice that reliably exhibited aggressive behaviors after consuming 1 g/kg of alcohol received systemic or intra-DRN administration of CRF-R1 antagonists, CP-154,526 or MTIP, before a confrontation with a male conspecific. Blockade of DRN CRF-R1 receptors with both antagonists significantly reduced only alcohol-heightened aggression, whereas systemic administration reduced both alcohol-heightened and species-typical aggression. Next, a 5-HT1A agonist, 8-OH-DPAT, was coadministered with CP-154,526 into the DRN to temporarily disrupt 5-HT activity. This manipulation abolished the antiaggressive effects of intra-DRN CP-154,526. In the mPFC, in vivo microdialysis revealed that extracellular 5-HT levels were increased in mice that consumed alcohol and were then injected with CP-154,526, both systemically or intra-DRN. Neither alcohol nor CP-154,526 alone affected 5-HT release in the mPFC. The present results suggest the DRN as a critical site for CRF-R1 to modulate alcohol-heightened aggression via action on the serotonergic DRN-PFC pathway.

Urinary exosome miRNome analysis and its applications to salt sensitivity of blood pressure.

OBJECTIVES: To investigate microRNAs (miRNAs) in urinary exosomes and their association with an individual's blood pressure response to dietary salt intake. DESIGN AND METHODS: Human urinary exosomal miRNome was examined by microarray. RESULTS: Of 1898 probes tested, 194 miRNAs were found in all subjects tested. 45 miRNAs had significant associations with salt sensitivity or inverse salt sensitivity. CONCLUSION: The expression of 45 urinary exosomal miRNAs associates with an individual's blood pressure response to sodium.