Valvular heart disease in Antiphospholipid antibody syndrome: Isolated Tricuspid stenosis

Antiphospholipid antibody syndrome (APLS) is a rare disorder characterized by a hypercoagulable state. Manifestations include arterial or venous thrombosis, recurrent fetal wastage, coronary artery disease, valvular heart disease, dilated cardiomyopathy, pulmonary artery hypertension, and intracardiac thrombus. Most commonly mitral valve is affected followed by aortic and then tricuspid valve. In this report, a rare case of spontaneous aortic thrombosis with tricuspid stenosis uncomplicated by other valve lesions is presented with clinical and echocardiographic studies and computed tomographic images.     

Malignant progression of B16 melanoma cells induced in vitro by growth factors produced by highly malignant cells

Four mouse B16 melanoma subclones (G3.15, G3.5, G3.12 and G3.26) exhibit progressively greater growth capacity in vitro and in vivo. Previously, non-metastatic G3.15 cells were sequentially converted, in monolayer cultures, to the moderately-metastatic G3.5 cells, and then to a highly-metastatic G3.5^* phenotype. Both conversions were induced by hypoxia followed by confluence, and also occurred in tumors. G3.5^* cells were comparable with, yet distinguishable from, G3.12 cells in being growth-autonomous in culture. In this study, the presumption that rapidly-growing G3.26 cells represented the ultimate progression step in this clonal system was examined. Both G3.12 and G3.5^* cells converted in vitro to the G3.26 phenotype during growth in serum-free medium conditioned by G3.26 cell growth. By selective filtration of conditioned medium and characterization of the stability of growth- and conversion-promoting activities, three distinct activities were found to promote a two-step G3.12 to G3.26 phenotype conversion: (1) a < 10 kDa filtrate stimulated slight attachment and proliferation of G3.12 cells, effects that were reversible, partly attributable to accumulated lactate, and fully mimicked by medium acidification to pH 6.5; (2) medium acidification, together with a heat- and acid-stable but partially trypsin-sensitive > 10 kDa activity, induced G3.12 G3.5^* conversion that resulted in acquisition of growth autonomy; and (3) a heat-, acid- and trypsin-sensitive > l0 kDa activity induced G3.5^* G3.26 conversion, characterized by anchorage-independent growth in soft agar, and potent lung colonization following intravenous injection. Phenotype analysis of G3.12 tumors and lung metastases revealed that G3.5^*-like cells were regularly present in tumors and metastases, whereas G3.26-like cells occurred almost exclusively in large lung metastases. While G3.12 cells might convert to G3.5^* cells in order to disseminate, G3.26 cells are apparently not involved in metastatic spread but probably account for the rapid growth of established metastases.     

Kinetics of the nitrogen dioxide initiated polymerization of acrylic acid

Acrylic acid (AA) was polymerized with NO₂ in tetrahydrofuran (THF) and in 1,4-dioxane. The effects of monomer and initiator concentration and of temperature on polymer conversion, initial rate of polymerization, and molecular weight were studied. The overall activation energy of polymerization was found to be 16,3 kcal mol^?1 (68,23 kJ · mol^?1) and 15,54 kcal · mol^?1 (65,05 kJ · mol^?1) in THF and in 1,4-dioxane, respectively. High molecular weight polymers (M ca. 10⁵) were obtained. The polymerization appears to be initiated by free radicals.     

Association of obesity with illness severity in hospitalized patients with COVID-19: A retrospective cohort study

BackgroundAlthough recent studies have shown an association between obesity and adverse coronavirus disease 2019 (COVID-19) patient outcomes, there is a paucity in large studies focusing on hospitalized patients. We aimed to analyze outcomes associated with obesity in a large cohort of hospitalized COVID-19 patients.MethodsWe performed a retrospective study at a tertiary care health system of adult patients with COVID-19 who were admitted between March 1 and April 30, 2020. Patients were stratified by body mass index (BMI) into obese (BMI ≥ 30 kg/m 2) and non-obese (BMI < 30 kg/m 2) cohorts. Primary outcomes were mortality, intensive care unit (ICU) admission, intubation, and 30-day readmission.ResultsA total of 1983 patients were included of whom 1031 (51.9%) had obesity and 952 (48.9%) did not have obesity. Patients with obesity were younger (P < 0.001), more likely to be female (P < 0.001) and African American (P < 0.001) compared to patients without obesity. Multivariable logistic models adjusting for differences in age, sex, race, medical comorbidities, and treatment modalities revealed no difference in 60-day mortality and 30-day readmission between obese and non-obese groups. In these models, patients with obesity had increased odds of ICU admission (adjusted OR, 1.37; 95% CI, 1.07?1.76; P = 0.012) and intubation (adjusted OR, 1.37; 95% CI, 1.04?1.80; P = 0.026).ConclusionsObesity in patients with COVID-19 is independently associated with increased risk for ICU admission and intubation. Recognizing that obesity impacts morbidity in this manner is crucial for appropriate management of COVID-19 patients.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins

The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ–20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.

Recent technological breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have greatly accelerated the pace of high-resolution structure determination of biological macromolecules. However, a major bottleneck in the structural study of important targets including membrane proteins, protein–DNA complexes, and large protein assemblies is sample production. The challenge of producing functional proteins extends beyond structural biology and impacts many areas of biological sciences that currently rely on techniques of protein overexpression. Conventional methods of protein production involve overexpression of a target gene from a plasmid within heterologous systems, including bacterial (Escherichia coli), insect (Sf9), and mammalian (HEK) cells. Overexpression approaches frequently encounter issues with proteins that are misfolded, nonfunctional, or degraded by the host cells. Overexpression of certain proteins may also alter protein homeostasis within cells. Substantial optimization is often necessary to identify the right conditions to produce suitable proteins for study, which is time-consuming and challenging in many cases. The problem is compounded further by multiprotein complexes where multiple subunits need to be assembled in the correct order and stoichiometries, making the coexpression of multiple subunits and purification of protein assemblies even more challenging.Studies of challenging proteins and large protein complexes have sometimes been possible by extracting samples from natural sources. While this approach has proven successful in special cases, examples of native proteins purified in this way have been largely limited to highly abundant and large protein assemblies that can be isolated by sucrose gradient ultracentrifugation (1) or where a known binding partner can be used as bait (2, 3). A general and efficient method to isolate proteins from endogenous sources with high specificity and at sufficient quantities for structural studies remains a goal of substantial interest. A rapid and efficient approach to add an affinity tag onto a target protein in human cells for affinity purification of endogenous complexes would greatly accelerate structural characterization of challenging and biomedically important targets. Furthermore, this strategy could be combined with complementary approaches such as mass spectrometry to better understand protein function under more native-like conditions.Recent technological advances in CRISPR-Cas–mediated genome engineering have provided new avenues to target endogenous proteins from native sources by genetically incorporating an affinity tag onto a protein of interest for purification and downstream analysis (4, 5). However, the time-consuming process of generating and selecting gene-edited cells with an endogenously tagged protein has created a substantial barrier for this approach to be widely adopted by the structural biology community and other fields of protein science. A simple, fast, and minimally perturbing way to target and extract endogenous proteins would make this approach more widely accessible to the general scientific community. Here we describe a simple and highly efficient method that leverages the unique properties of split fluorescent proteins (6) to select cells harboring an affinity tag on target endogenous proteins for purification.We demonstrate this approach on the study of the human proteasome. The proteasome plays an essential role in protein degradation and is critical to maintaining cellular protein homeostasis (7). Changes to proteostasis in cells and lowered protein degradation due to decreases in proteasomal activity have been linked to aging and neurodegeneration (8). While the proteasome has been a topic of extensive studies using a variety of strategies (9–12), the study of some human proteasomal complexes has remained challenging. In this study, by tagging, purifying, and determining structures of various human proteasomal complexes from HEK cells we demonstrate that the efficient protein tagging method presented here can facilitate rapid structural studies of endogenous human protein complexes in HEK cells. Analyzing cryo-EM structures of PA28–20S complexes reveals insights into PA28 stoichiometry and sheds light on how unfolded polypeptide substrates traverse through the antechamber of the 20S core particle before entering the proteolytic chamber for degradation.     

Dyke-Davidoff-Masson syndrome: A rare case of hemiatrophy of brain—Case report from Nepal

Dyke-Davidoff-Masson syndrome (DDMS) is a rare neurological disorder that results from brain injury during intrauterine or early years of life. Prominent cortical sulci, dilated lateral ventricles, cerebral hemiatrophy, hyperpneumatization of the sinus, and compensatory hypertrophy of the skull are the characteristic findings. We describe a female patient who presented with a history of seizure, right-sided body weakness, and neuroimaging features of left cerebral hemiatrophy, dilatation of left lateral ventricle, left frontal sinus hyperpneumatization, asymmetric calvarial thickening, and elevation of the petrous ridge.     

Reverse Swiss Roll technique for diabetic vitreous membranes

Background:Diabetic membranes are always a challenge for a surgeon because of sticky nature and chances of iatrogenic break while removing.Purpose:To demonstrate a safe reverse swiss roll technique to dissect diabetic vitreous membranes.Synposis:Approaches and techniques for membrane dissection are segmentation, delamination and en-bloc dissection using various types of instruments and illumination. With vitreous cutte, picks and scissors, surgical steps are traditionallu performed by using classic lift and shave technique. After identifying the plane, tissue is lifted and then cut, which puts the retina at risk of break because of traction and active suction. Such a threat can be reduced by placing the cutter above the membrane thereby having the membrane itseld acting as a protective cushion to the retina. Port here, unlike lift and shave technique, doesn’t face the edge of membrane but is exactly 180 degree opposite and membrane curls into port because of suction. Also, hemostasis is maintained by continuous aspiration and cutting as the instrument is moved side to side, retracting from the edge.Highlights:Reverse swiss roll technique is safer compared to lift and shave because of the safety cushion of the membrane between the port and the retina. There is inherently less chances of retinal break because the active suction from the port is directed away from the retina. The technique also minimises traction and localised pull on the atrophic macula.Video link: https://youtu.be/WNnSsP69ZLw     

The ALK inhibitor ASP3026 eradicates NPM-ALK+ T-cell anaplastic large-cell lymphoma in vitro and in a systemic xenograft lymphoma model

NPM-ALK⁺ T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer. Standard treatment of NPM-ALK⁺ ALCL is CHOP polychemotherapy. Although patients initially respond favorably to CHOP, resistance, relapse, and death frequently occur. Recently, selective targeting of ALK has emerged as an alternative therapeutic strategy. ASP3026 is a second-generation ALK inhibitor that can overcome crizotinib resistance in non-small cell lung cancer, and is currently being evaluated in clinical trials of patients with ALK⁺ solid tumors. However, NPM-ALK⁺ ALCL patients are not included in these trials. We studied the effects of ASP3026 on NPM-ALK⁺ ALCL cell lines in vitro and on systemic lymphoma growth in vivo. ASP3026 decreased the viability, proliferation, and colony formation, as well as induced apoptotic cell death of NPM-ALK⁺ ALCL cells. In addition, ASP3026 significantly reduced the proliferation of 293T cells transfected with NPM-ALK mutants that are resistant to crizotinib and downregulated tyrosine phosphorylation of these mutants. Moreover, ASP3026 abrogated systemic NPM-ALK⁺ ALCL growth in mice. Importantly, the survival of ASP3026-treated mice was superior to that of control and CHOP-treated mice. Our data suggest that ASP3026 is an effective treatment for NPM-ALK⁺ ALCL, and support the enrollment of patients with this lymphoma in the ongoing clinical trials.