Inverted triplications formed by iterative template switches generate structural variant diversity at genomic disorder loci.

The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a complex genomic rearrangement (CGR). Although it has been identified as an important pathogenic DNA mutation signature in genomic disorders and cancer genomes, its architecture remains unresolved. Here, we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the DNA of 24 patients identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted structural variant (SV) haplotypes. Using a combination of short-read genome sequencing (GS), long-read GS, optical genome mapping, and single-cell DNA template strand sequencing (strand-seq), the haplotype structure was resolved in 18 samples. The point of template switching in 4 samples was shown to be a segment of ∼2.2-5.5 kb of 100% nucleotide similarity within inverted repeat pairs. These data provide experimental evidence that inverted low-copy repeats act as recombinant substrates. This type of CGR can result in multiple conformers generating diverse SV haplotypes in susceptible dosage-sensitive loci.

Mind the gap: the relevance of the genome reference to resolve rare and pathogenic inversions.

Both long-read genome sequencing (lrGS) and the recently published Telomere to Telomere (T2T) reference genome provide increased coverage and resolution across repetitive regions promising heightened structural variant detection and improved mapping. Inversions (INV), intrachromosomal segments which are rotated 180° and inserted back into the same chromosome, are a class of structural variants particularly challenging to detect due to their copy-number neutral state and association with repetitive regions. Inversions represent about 1/20 of all balanced structural chromosome aberrations and can lead to disease by gene disruption or altering regulatory regions of dosage sensitive genes in cis . Here we remapped the genome data from six individuals carrying unsolved cytogenetically detected inversions. An INV6 and INV10 were resolved using GRCh38 and T2T-CHM13. Finally, an INV9 required optical genome mapping, de novo assembly of lrGS data and T2T-CHM13. This inversion disrupted intron 25 of EHMT1, confirming a diagnosis of Kleefstra syndrome 1 (MIM#610253). These three inversions, only mappable in specific references, prompted us to investigate the presence and population frequencies of differential reference regions (DRRs) between T2T-CHM13, GRCh37, GRCh38, the chimpanzee and bonobo, and hundreds of megabases of DRRs were identified. Our results emphasize the significance of the chosen reference genome and the added benefits of lrGS and optical genome mapping in solving rearrangements in challenging regions of the genome. This is particularly important for inversions and may impact clinical diagnostics.

Bunch of grapes - I saw the sign.

As early as the late 1800s, the appearance of hydatidiform moles have been likened to grapes. The bunch of grapes sign is an ultrasound finding of a heterogenous mass within the uterine cavity with multiple anechoic spaces. We saw the bunch of grapes sign on a 28-year-old on ultrasound.

Single-Cell Classification Based on Population Nucleus Size Combining Microwave Impedance Spectroscopy and Machine Learning.

Many recent efforts in the diagnostic field address the accessibility of cancer diagnosis. Typical histological staining methods identify cancer cells visually by a larger nucleus with more condensed chromatin. Machine learning (ML) has been incorporated into image analysis for improving this process. Recently, impedance spectrometers have been shown to generate all-inclusive lab-on-a-chip platforms to detect nucleus abnormities. In this paper, a wideband electrical sensor and data analysis paradigm that can identify nuclear changes shows the realization of a single-cell microfluidic device to detect nuclei of altered sizes. To model cells of altered nucleus, Jurkat cells were treated to enlarge or shrink their nucleus followed by broadband sensing to obtain the S-parameters of single cells. The ability to deduce important frequencies associated with nucleus size is demonstrated and used to improve classification models in both binary and multiclass scenarios, despite a heterogeneous and overlapping cell population. The important frequency features match those predicted in a double-shell circuit model published in prior work, demonstrating a coherent new analytical technique for electrical data analysis. The electrical sensing platform assisted by ML with impressive accuracy of cell classification looks forward to a label-free and flexible approach to cancer diagnosis.

Postpartum left circumflex to left atrial fistula: A rare presentation.

Coronary artery fistulas (CAFs) are rare and often asymptomatic, but severe complications can occur, resulting in heart failure and cardiac arrhythmia. They have been associated with iatrogenic or traumatic injuries as well as systemic inflammatory conditions. However, there have been very few documented cases of pregnancy associated CAFs. We observed a case of left circumflex to left atrium fistula in a 37-year-old female presenting with insidious onset of progressive dyspnea during pregnancy.

The Association Between Mood and Anxiety Disorders With Perioperative Outcomes Following Radical Cystectomy.

OBJECTIVE: To identify associations between preoperative psychiatric diagnoses and perioperative outcomes after RC. METHODS: The Florida Inpatient Data File was used to identify patients who underwent RC from 2013 to 2019. ICD-10 codes for a mood or anxiety disorder were identified and analyzed as a 3-level variable: neither, one of these, or both. Outcomes included inpatient mortality, non-home discharge, in-hospital complications, and length of stay. Mixed-effects logistic regression (accounting for clustering within hospitals) and negative binomial regression models were utilized. RESULTS: We identified 4396 RC patients, including 306 (7.0%) with a mood disorder and 389 (8.8%) with an anxiety disorder. After multivariable adjustment, there was no significant association between mood and/or anxiety disorders with mortality or the presence or number of in-hospital complications. However, a mood or anxiety disorder was significantly associated with increased odds of non-home discharge (OR 1.60, 95% CI 1.20-2.14) and longer length of stay (IRR 1.13, 95% CI 1.07-1.19); these associations were also increased among patients with both mood and anxiety disorder diagnoses (non-home discharge OR 2.66, 95% CI 1.61-4.38; length of stay IRR 1.12, 95% CI 1.01-1.24). CONCLUSION: Patients with mood and/or anxiety disorders undergoing RC had longer length of stay and increased odds of discharge to a non-home facility despite similar risks of perioperative complications. These data suggest an opportunity for perioperative intervention to address these disparities in postoperative outcomes. However, further work is needed to determine the underlying causes of these differences and to develop effective interventions.

Trifocal Tibial Bone Transport Using a Magnetic Intramedullary Nail: A Case Report.

CASE: The patient sustained a pathologic fracture through chronic osteomyelitis of the tibia. Treatment involved resection of bone and soft tissue, leaving a 15-cm diaphyseal tibial defect. We designed a construct to allow for trifocal bone transport using a magnetic intramedullary lengthening nail connected to cables. This technique uses cables secured to the proximal tibia, the nail, and the transport segments. CONCLUSION: This case details the successful use of trifocal cable-assisted bone transport through a magnetic lengthening intramedullary nail. This technique can successfully be used to reconstruct diaphyseal bone defects that are longer than the stroke length of the magnetic nail.

Broadband electrical impedance as a novel characterization of oxidative stress in single L6 skeletal muscle cells.

Oxidative stress (OS) is one of the leading causes of cytotoxicity and is linked to many human physio-pathological conditions. In particular, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) induced by OS is debilitating to quality of life, while no clear biological markers have been identified for diagnostic measures. Recently, impedance measurements of peripheral blood cells of ME/CFS patients have been shown as a promising approach to diagnose the disease. Inspired by this study and aiming to interrogate muscle cells directly, we investigated if broadband measurements of single muscle cells could differentiate normal and oxidatively stressed cell populations. We first optimized a protocol through H2O2 treatment to introduce oxidative stress to cultured rat L6 skeletal muscle cells. The treated cells were further characterized through broadband impedance spectroscopy of single cells using a microfluidic lab-on-a-chip system. The resulting dielectric properties of cytoplasm permittivity and conductivity are electrically distinct from normally cultured cells. The reflection and transmission coefficients, ΔS11 and ΔS21, of the normal cells are tightly clustered and closely resemble those of the cell-free solution across the frequency range of 9 kHz to 9 GHz. On the other hand, dielectric properties of the oxidized cells have a wide distribution in the GHz range, deviating both in the positive and negative directions from the normally cultured cells. Simulation results guide our hypothesis that the dielectric differences could be linked to ion alterations, while calcium imaging directly supports the contribution of calcium flux to the observed deviation of S parameters. The unique electrical profile associated with oxidized cells in the GHz frequencies provide a framework for future development of technologies to diagnose oxidative-stress related diseases such as ME/CFS.

Broadband Electrical Sensing of a Live Biological Cell with In Situ Single-Connection Calibration.

Single-connection in situ calibration using biocompatible solutions is demonstrated in single-cell sensing from 0.5 to 9 GHz. The sensing is based on quickly trapping and releasing a live cell by dielectrophoresis on a coplanar transmission line with a little protrusion in one of its ground electrodes. The same transmission line is used as the calibration standard when covered by various solutions of known permittivities. The results show that the calibration technique may be precise enough to differentiate cells of different nucleus sizes, despite the measured difference being less than 0.01 dB in the deembedded scattering parameters. With better accuracy and throughput, the calibration technique may allow broadband electrical sensing of live cells in a high-throughput cytometer.

Early comprehensive testing for COVID-19 is essential to protect trauma centers.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presents a threat to health care systems worldwide. Trauma centers may be uniquely impacted, given the need for rapid invasive interventions in severely injured and the growing incidence of community infection. We discuss the impact that SARS-CoV-2 has had in our trauma center and our steps to limit the potential exposures. METHODS: We performed a retrospective evaluation of the trauma service, from March 16 to 30, following the appearance of SARS-CoV-2 in our state. We recorded the daily number of trauma patients diagnosed with SARS-CoV-2 infection, the presence of clinical symptoms or radiological signs of COVID-19, and the results of verbal symptom screen (for new admissions). The number of trauma activations, admissions, and census, as well as staff exposures and infections, was recorded daily. RESULTS: Over the 14-day evaluation period, we tested 85 trauma patients for SARS-CoV-2 infection, and 21 (25%) were found to be positive. Sixty percent of the patients in the trauma/burn intensive care unit were infected with SARS-CoV-2. Positive verbal screen results, presence of ground glass opacities on admission chest CT, and presence of clinical symptoms were not significantly different in patients with or without SARS-CoV-2 infection (p > 0.05). Many infected patients were without clinical symptoms (9/21, 43%) or radiological signs on admission (18/21, 86%) of COVID-19. CONCLUSION: Forty-five percent of trauma patients are asymptomatic at the time of SARS-CoV-2 diagnosis. Respiratory symptoms, as well as verbal screening (recent fevers, shortness of breath, cough, international travel, and close contact with known SARS-CoV-2 carriers), are inaccurate in the trauma population. These findings demonstrate the need for comprehensive rapid testing of all trauma patients upon presentation to the trauma bay. LEVEL OF EVIDENCE: Diagnostic tests or criteria, level III, Therapeutic/care management, level IV.

Notochord vacuoles absorb compressive bone growth during zebrafish spine formation.

The vertebral column or spine assembles around the notochord rod which contains a core made of large vacuolated cells. Each vacuolated cell possesses a single fluid-filled vacuole, and loss or fragmentation of these vacuoles in zebrafish leads to spine kinking. Here, we identified a mutation in the kinase gene dstyk that causes fragmentation of notochord vacuoles and a severe congenital scoliosis-like phenotype in zebrafish. Live imaging revealed that Dstyk regulates fusion of membranes with the vacuole. We find that localized disruption of notochord vacuoles causes vertebral malformation and curving of the spine axis at those sites. Accordingly, in dstyk mutants the spine curves increasingly over time as vertebral bone formation compresses the notochord asymmetrically, causing vertebral malformations and kinking of the axis. Together, our data show that notochord vacuoles function as a hydrostatic scaffold that guides symmetrical growth of vertebrae and spine formation.

Black Phosphorus High-Frequency Transistors with Local Contact Bias.

Having a sizable band gap and high carrier mobility, black phosphorus (BP) is a promising two-dimensional material for high-frequency electronic and optoelectronic devices. Further, for metal-oxide-semiconductor field-effect transistors (MOSFETs) operating at high frequencies, they must have a top gate of submicron length instead of the commonly used global back gate. However, without the global back gate to electrostatically induce doping in BP, top-gated submicron BP MOSFETs have not reached their full potential mainly due to large contact resistances. Here, we report top-gated submicron BP MOSFETs with local contact bias electrodes to induce doping in the contact region. This resulted in reduced contact resistance and, in turn, orders of magnitude improvement in current capacity (>500 µA/µm) and peak transconductance (>40 µS/µm), if compared with top-gated BP transistors without any back-gating scheme. In turn, these improvements resulted in a forward current gain cutoff frequency of 37 GHz and a maximum frequency of oscillation of 22 GHz at room temperature, the highest reported for BP MOSFETs up to date.

Correlation Between Optical Fluorescence and Microwave Transmission During Single-Cell Electroporation.

OBJECTIVE: Multimodal characterization of a mammalian cell by optical and microwave techniques simultaneously during electroporation. METHODS: Using a coplanar waveguide with a Jurkat cell trapped in the middle of its center conductor, continuous waves at 100 kHz of different amplitudes were applied for 20 s, while microwave transmission coefficients at 9 GHz were measured every 0.4 s. RESULTS: The onset of electroporation was indicated by abrupt changes in both fluorescence intensity and transmission coefficient. Additionally, in measurements that lasted 300 s, the transmission coefficient was found to recover to the pre-poration level, while the fluorescence intensity remained different. Since the cells were confirmed viable through post-poration staining, the recovery of the transmission coefficient suggested reversible electroporation. CONCLUSION: These experimental results showed that the transmission coefficient could serve as a label-free indicator of cell membrane permeability during and after electroporation. Furthermore, it could be used to expeditiously differentiate reversible electroporation from the irreversible one. SIGNIFICANCE: This study should aid fundamental analysis of cell physiology, as well as molecular delivery, in cell engineering and electrotherapy.

A Comparative Study of the ReCell® Device and Autologous Spit-Thickness Meshed Skin Graft in the Treatment of Acute Burn Injuries.

Early excision and autografting are standard care for deeper burns. However, donor sites are a source of significant morbidity. To address this, the ReCell® Autologous Cell Harvesting Device (ReCell) was designed for use at the point-of-care to prepare a noncultured, autologous skin cell suspension (ASCS) capable of epidermal regeneration using minimal donor skin. A prospective study was conducted to evaluate the clinical performance of ReCell vs meshed split-thickness skin grafts (STSG, Control) for the treatment of deep partial-thickness burns. Effectiveness measures were assessed to 1 year for both ASCS and Control treatment sites and donor sites, including the incidence of healing, scarring, and pain. At 4 weeks, 98% of the ASCS-treated sites were healed compared with 100% of the Controls. Pain and assessments of scarring at the treatment sites were reported to be similar between groups. Significant differences were observed between ReCell and Control donor sites. The mean ReCell donor area was approximately 40 times smaller than that of the Control (P < .0001), and after 1 week, significantly more ReCell donor sites were healed than Controls (P = .04). Over the first 16 weeks, patients reported significantly less pain at the ReCell donor sites compared with Controls (P ≤ .05 at each time point). Long-term patients reported higher satisfaction with ReCell donor site outcomes compared with the Controls. This study provides evidence that the treatment of deep partial-thickness burns with ASCS results in comparable healing, with significantly reduced donor site size and pain and improved appearance relative to STSG.

Measuring zinc in biological nanovesicles by multiple analytical approaches.

Exosomes are nanovesicles known to mediate intercellular communication. Although it is established that zinc ions can act as intracellular signaling factors, the measurement of zinc in circulating nanovesicles has not yet been attempted. Providing evidence of the existence of this zinc fraction and methods for its measurement might be important to advance our knowledge of zinc status and its relevance in diseases. Exosomes from 0.5 ml of either fresh or frozen human plasma were isolated by differential centrifugation. A morphological and dimensional evaluation at the nanoscale level was performed by atomic force microscopy (AFM) and Transmission Electron Microscopy (TEM). Energy Dispersive X-Ray Microanalysis (EDX) revealed the elemental composition of exosomes and their respective total Zinc content on a quantitative basis. The zinc mole fraction (in at%) was correlated to the phosphorous mole fraction, which is indicative for exosomal membrane material. Both fresh (Zn/P 0.09 ±â€¯0.01) and frozen exosomes (Zn/P 0.08 ±â€¯0.02) had a significant zinc content, which increased up to 1.09 ±â€¯0.12 for frozen exosomes when treated with increasing amounts of zinc (100-500 µM; each p < 0.05). Interestingly, after zinc addition, the Calcium mole fractions decreased accordingly suggesting a possible exchange by zinc. In order to estimate the intra-exosomal labile zinc content, an Imaging Flow Cytometry approach was developed by using the specific membrane permeable zinc-probe Fluozin-3AM. A labile zinc content of 0.59 ±â€¯0.27 nM was calculated but it is likely that the measurement may be affected by purification and isolation conditions. This study suggests that circulating nano-vesicular-zinc can represent a newly discovered zinc fraction in the blood plasma whose functional and biological properties will have to be further investigated in future studies.

Hypertrophic Scar Severity at Autograft Sites Is Associated With Increased Pain and Itch After Major Thermal Burn Injury.

Approximately three quarters of major thermal burn injury (MThBI) survivors suffer from hypertrophic scarring (HTS) and over half experience chronic pain or itch. In survivors of MThBI, HTS and chronic pain or itch are considered one of the greatest unmet challenges of postburn injury care and psychosocial reintegration. Although scarring, itch, and pain have been clinically associated, there are no prospective, multisite studies examining tissue autograft site pain or itch and scar outcomes. The authors collected a representative cohort (n = 56) of MThBI survivors who received autografting within 14 days of injury and evaluated graft-site pain or itch severity (0-10 Numeric Rating Scale) and HTS using a validated scar photograph assessment scale 6 months following MThBI. Given that stress is known to influence wound healing, the authors also assessed the relationship between previous trauma exposure, peritraumatic stress, preburn overall health (SF-12), scarring, and chronic pain or itch severity using Spearman's correlation. Association between HTS and chronic pain or itch was significant in a linear regression model adjusted for age, sex, and ethnicity (ß = 0.2, P = .033 for pain, ß = 0.2, P = .019 for itch). Results indicate that prior trauma exposure is inversely correlated (r = -.363, P = .030) with scar severity, but not pain or itch severity 6 months after MThBI. Study results suggest that preburn chronic pain or itch is associated with pathological scarring 6 months following MThBI. Results also indicate that stress may improve scarring after MThBI. Further work to understand the mechanisms that underlie both HTS and chronic pain or itch and their relationship to chronic stress is critical to the development of novel therapies to assist burn survivors recover.

Pain and itch outcome trajectories differ among European American and African American survivors of major thermal burn injury.

More than half of individuals experiencing major thermal burn injury (MThBI) receive an autologous skin graft (autograft), in which skin is removed from a healthy "donor" site and transplanted to the burn site. Persistent pain and itch at the graft site are major causes of suffering and disability in MThBI survivors. African Americans have a higher risk of MThBI, and in other clinical settings African Americans experience a greater burden of pain and itch relative to European Americans. However, to our knowledge, ethnic differences in skin graft site pain and itch outcomes after MThBI have not been assessed. We evaluated skin graft site pain and itch severity (0-10 Numeric Rating Scale [NRS]) over 1 year in a prospective multicenter cohort sample of African Americans and European Americans. In adjusted linear mixed models, African Americans experienced a slower rate of pain resolution in the acute phase of recovery (ß = -0.05 vs -0.08 NRS points per day, P < 0.001), which resulted in a higher pain severity in the persistent phase of recovery (NRS mean difference = 1.21, 95% confidence interval [0.12-2.29]), although not statistically significant after correction for multiple comparisons. African Americans also experience greater itch severity in 6 weeks to 12 months after burn injury compared with European Americans (NRS mean difference = 1.86 [0.80-2.93]), which results from a faster rate of itch development in African Americans in the acute recovery phase after burn injury. Future studies may improve outcomes in African Americans and lead to new pathogenic insights that benefit all burn injury survivors.

Chronic Pain and Itch are Common, Morbid Sequelae Among Individuals Who Receive Tissue Autograft After Major Thermal Burn Injury.

OBJECTIVE: Pain and itch symptoms are common after major thermal burn injury (MThBI)-requiring tissue autografting. To our knowledge, no prospective longitudinal studies have characterized pain and itch outcomes after tissue autografting and associations between and functional interference caused by such symptoms. MATERIALS AND METHODS: We prospectively evaluated burn graft site and tissue donor site pain and itch severity (0 to 10, numeric rating scale) over 1 year among a representative cohort of MThBI survivors (n=96) who received tissue autografting within 14 days of MThBI. RESULTS: Nearly all participants had moderate or severe burn pain at the time of enrollment. Most individuals experienced an upper extremity burn with donor tissue taken from thigh. Persistent moderate or severe burn graft site pain declined thereafter, but remained common, with 25/90 (28%), 24/77 (31%), and 17/82 (21%) experiencing moderate or severe pain at 6 weeks, 3 months, and 6 months, respectively. Although there was improved function after immediate postinjury decline in all participants, those who had moderate or severe pain showed worse functional outcomes at each timepoint. Significant correlations were present between itch and pain burden over time at the same site (ie, autograft site r=0.629, P<0.01) and also across sites (ie, autograft and donor site itch r=0.552, P<0.01). DISCUSSION: Pain and itch are common after MThBI, are temporally and spatially concordant and cause significant impact on daily function. Further studies are needed to better understand pain and itch symptom pathogenesis after MThBI, to reduce the tremendous suffering and decline.

A UBE2O-AMPKα2 Axis that Promotes Tumor Initiation and Progression Offers Opportunities for Therapy.

UBE2O is localized in the 17q25 locus, which is known to be amplified in human cancers, but its role in tumorigenesis remains undefined. Here we show that Ube2o deletion in MMTV-PyVT or TRAMP mice profoundly impairs tumor initiation, growth, and metastasis, while switching off the metabolic reprogramming of tumor cells. Mechanistically, UBE2O specifically targets AMPKα2 for ubiquitination and degradation, and thereby promotes activation of the mTOR-HIF1α pathway. Notably, inactivation of AMPKα2, but not AMPKα1, abrogates the tumor attenuation caused by UBE2O loss, while treatment with rapamycin or inhibition of HIF1α ablates UBE2O-dependent tumor biology. Finally, pharmacological blockade of UBE2O inhibits tumorigenesis through the restoration of AMPKα2, suggesting the UBE2O-AMPKα2 axis as a potential cancer therapeutic target.