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We show low-cost fabrication and characterization of borosilicate glass nanopores for single molecule sensing. Nanopores with diameters of ~100 nm were fabricated in borosilicate glass capillaries using laser assisted glass puller. We further achieve controlled reduction and nanometer-size control in pore diameter by sculpting them under constant electron beam exposure. We successfully fabricate pore diameters down to 6 nm. We next show electrical characterization and low-noise behavior of these borosilicate nanopores and compare their taper geometries. We show, for the first time, a comprehensive characterization of glass nanopore conductance across six-orders of magnitude (1M-1μM) of salt conditions, highlighting the role of buffer conditions. Finally, we demonstrate single molecule sensing capabilities of these devices with real-time translocation experiments of individual λ-DNA molecules. We observe distinct current blockage signatures of linear as well as folded DNA molecules as they undergo voltage-driven translocation through the glass nanopores. We find increased signal to noise for single molecule detection for higher trans-nanopore driving voltages. We propose these nanopores will expand the realm of applications for nanopore platform.

Background As providers look to scale high-acuity care in the patient home setting, hospital-at-home is becoming more prevalent. The traditional model of hospital-at-home usually relies on care delivery by in-home providers, caring for patients in urban communities through academic medical centers. Our objective is to describe the process and outcomes of Mayo Clinic’s Advanced Care at Home (ACH) program, a hybrid virtual and in-person hospital-at-home model combining a single, virtual provider-staffed command center with a vendor-mediated in-person medical supply chain to simultaneously deliver care to patients living near an urban hospital-at-home command center and patients living in a rural region in a different US state and time zone. Methods A descriptive, retrospective medical records review of all patients admitted to ACH between July 6, 2020, and December 31, 2021. Patients were admitted to ACH from an urban academic medical center in Florida and a rural community hospital in Wisconsin. We collected patient volumes, age, sex, race, ethnicity, insurance type, primary hospital diagnosis, 30-day mortality rate, in-program mortality, 30-day readmission rate, rate of return to hospital during acute phase, All Patient Refined-Diagnosis Related Groups (APR-DRG) Severity of Illness (SOI), and length of stay (LOS) in both the inpatient-equivalent acute phase and post-acute equivalent restorative phase. Results Six hundred and eighty-six patients were admitted to the ACH program, 408 in Florida and 278 in Wisconsin. The most common diagnosis seen were infectious pneumonia (27.0%), septicemia / bacteremia (11.5%), congestive heart failure exacerbation (11.5%), and skin and soft tissue infections (6.3%). Median LOS in the acute phase was 3 days (IQR 2–5) and median stay in the restorative phase was 22 days (IQR 11–26). In-program mortality rate was 0% and 30-day mortality was 0.6%. The mean APR-DRG SOI was 2.9 (SD 0.79) and the 30-day readmission rate was 9.7%. Conclusions The ACH hospital-at-home model was able to provide both high-acuity inpatient-level care and post-acute care to patients in their homes through a single command center to patients in urban and rural settings in two different geographical locations with favorable outcomes of low mortality and hospital readmissions.

The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications. CRISPR–Cas9, when loaded with the guide RNA, cleaves the DNA at the target position as recognized by the guide RNA sequence. For successful application of this technology, it is important to study the biophysical parameters affecting its function. Temperature dependence of the Cas9 binding as well as energetics of product release after cleavage has not been well reported in the literature. In this work, we study the binding properties of Cas9 enzyme to the sequence specific target DNA at a range of temperatures and, surprisingly, find that the Cas9 enzyme, in our study, can find and bind its target DNA with 90 ± 20% efficiency at temperatures as low as 4 °C. Further, we show that the cleaved DNA products remain bound to the Cas9 enzyme strongly and is released from the enzyme only at higher temperatures. Using the gel shift assays, we quantify the rate of Cas9 binding to target DNA to be 0.8 ± 0.2 min −1 at 37 °C. We also tested denaturant (SDS) dependent release of cleaved product which showed a similar release pattern with a dissociation constant of 0.23 ± 0.04 mM. Our results of heat and denaturant dependence on Cas9–DNA binding and release mechanics will provide valuable insights for developing temperature dependent applications of the CRISPR–Cas9 technology.

Purpose. To evaluate the clinical and ultrasonographic changes in the morphology and vascularity of the common extensor tendon after injecting platelet-rich plasma (PRP) or corticosteroid (CS) for recalcitrant lateral epicondylitis (LE). Methods. 30 patients aged 18 to 60 years with recalcitrant (>6 months) LE not responsive to oral medication or non-invasive treatment were randomised to receive PRP (n=15) or CS (n=15) injection. Patients were assessed using the visual analogue scale (VAS) for pain, Disabilities of the Arm, Shoulder and Hand Scale (DASH) score, Oxford Elbow Score, modified Mayo Clinic performance index for the elbow (modified Mayo score), and hand grip strength. Ultrasonography was performed by a musculoskeletal ultrasonologist to evaluate for tear at the common extensor origin, oedema at the common extensor origin, cortical erosion, probe-induced tenderness, and thickness of the tendon. Results. The VAS for pain, DASH score, Oxford Elbow Score, modified Mayo score, and hand grip strength all improved significantly from pre-injection to the 6-month follow-up in the PRP and CS groups. However, in the CS group, the scores generally peaked at 3 months and then deteriorated slightly at 6 months indicating recurrence of symptoms, which involved 46.7% of the CS patients. At 6 months, the number of patients positive for various ulrasonographic findings generally decreased. However, in the CS group, the number of patients with reduced thickness of the common extensor tendon increased from 2 to 12, and the number of patients with cortical erosion at the lateral epicondyle increased from 9 to 11. Conclusion. PRP appeared to enable biological healing of the lesion, whereas CS appeared to provide short-term, symptomatic relief but resulted in tendon degeneration.

Chronic heart failure (CHF) remains the only cardiovascular disease with an increasing hospitalization burden and an ongoing drain on health care expenditures. The prevalence of CHF increases with advancing life span, with diastolic heart failure predominating in the elderly population. Primary prevention of coronary artery disease and risk factor management via aggressive blood pressure control are central in preventing new occurrences of left ventricular dysfunction. Optimal therapy for CHF involves identification and correction of potentially reversible precipitants, target-dose titration of medical therapy, and management of hospitalizations for decompensation. The etiological phenotype, absolute decrease in left ventricular ejection fraction and a widening of QRS duration on electrocardiography, is commonly used to identify patients at increased risk of progression of heart failure and sudden death who may benefit from prophylactic implantable cardioverter-defibrillator placement with or without cardiac resynchronization therapy. Patients who transition to advanced stages of disease despite optimal traditional medical and device therapy may be candidates for hemodynamically directed approaches such as a left ventricular assist device; in selected cases, listing for cardiac transplant may be warranted.

Nanopore sensing, a high-resolution DNA sequencing technology, is rapidly expanding into novel and exciting directions of probing specific DNA-enzyme interactions. Although proven excellent for the detecting structural features of bare DNA, quantitative measurements on enzyme-DNA complexes and their real-time activity are lagging and only starting to emerge for long DNA templates. Signal-to-noise requirement and high translocation speeds make it difficult to detect protein bound on biologically relevant plasmid-length DNA. To this end we report accurate position detection of a catalytically active Cas9 bound to its single or multiple target sites on the DNA. Protein position is fingerprinted using event charge deficit (ECD) based analysis of the high signal-to-noise electrical signals as the complex translocates through a glass nanopore. Using a time-dependent assay, we quantify the kinetics of the released products upon enzymatic cleavage of the target DNA by the wild-type Cas9 nuclease. Our approach enables the nanopore-based single-molecule sensing of DNA-protein complexes, for real-time monitoring of biochemical reactions. This may help understand protein binding & localization as well as improve Cas9-based targeting in genome engineering applications. Graphical abstract

Background In July 2020, Mayo Clinic launched Advanced Care at Home (ACH), a high-acuity virtual hybrid hospital-at-home model (HaH) of care at Mayo Clinic Florida and Northwest Wisconsin, an urban destination medical center and a rural community practice respectively. This study aims to describe demographic characteristics of ACH patients as well as their acuity of illness using severity of illness (SOI) and risk of mortality (ROM), to illustrate the complexity of patients in the program, taking into account the different diagnostic related groups. Methods Mayo Clinic uses All Patient Refined-Diagnosis Related Groups (APR-DRG) to calculate SOI and ROM on hospitalized patients. APR-DRG data, including SOI and ROM, were gathered from individual chart reviews from July 6, 2020, to March 31, 2022. Results  Out of 923 patients discharged from ACH, the average APR-DRG SOI was 2.89 (SD 0.81) and ROM was 2.73. (SD 0.92). Mean age was 70.88 (SD 14.46) years, 54.6% were male patients and the average length of stay was 4.10 days. The most frequent diagnosis was COVID-19 infection with 162 patients (17.6%), followed by heart failure exacerbation (12.7%) and septicemia (10.9%). The 30-day readmission rate after discharge from ACH was 11.2% ( n  = 103) and the 30-day mortality rate was 1.8% ( n  = 17). There were no in-program patient deaths. Conclusions SOI and ROM from patients at the ACH program have been shown to be in the range of “moderate/major” according to the APR-DRG classification. The ACH program is capable of accepting and managing highly complex patients that require advanced therapeutic means. Furthermore, the ACH program has an in-program mortality rate of 0 to date. Therefore, ACH is rising as a capable alternative to the brick-and-mortar hospital.

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for ∼$1,000 in ∼24 h.

Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis. High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes.

The current COVID-19 crisis has significantly impacted healthcare systems worldwide. There has been a palpable increase in public avoidance of hospitals, which has interfered in timely care of critical cardiovascular conditions. Complications from late presentation of myocardial infarction, which had become a rarity, resurfaced during the pandemic. We present two such encounters that occurred due to delay in seeking medical care following myocardial infarction due to the fear of contracting COVID-19 in the hospital. Moreover, a comprehensive review of literature is performed to illustrate the potential factors delaying and decreasing timely presentations and interventions for time-dependent medical emergencies like ST-segment elevation myocardial infarction (STEMI). We emphasise that clinicians should remain vigilant of encountering rare and catastrophic complications of STEMI during this current era of COVID-19 pandemic.