Structures of the junctophilin/voltage-gated calcium channel interface reveal hot spot for cardiomyopathy mutations.

SignificanceIon channels have evolved the ability to communicate with one another, either through protein-protein interactions, or indirectly via intermediate diffusible messenger molecules. In special cases, the channels are part of different membranes. In muscle tissue, the T-tubule membrane is in proximity to the sarcoplasmic reticulum, allowing communication between L-type calcium channels and ryanodine receptors. This process is critical for excitation-contraction coupling and requires auxiliary proteins like junctophilin (JPH). JPHs are targets for disease-associated mutations, most notably hypertrophic cardiomyopathy mutations in the JPH2 isoform. Here we provide high-resolution snapshots of JPH, both alone and in complex with a calcium channel peptide, and show how this interaction is targeted by cardiomyopathy mutations.

Management of donor kidneys with double renal arteries with significant luminal discrepancy: A retrospective cohort study.

INTRODUCTION: Side-to-side pantaloon anastomosis for renal grafts with double renal arteries (RA) with significant luminal discrepancy between graft arteries has not been reported. We hypothesized that the pantaloon technique is feasible and safe in these cases. MATERIALS AND METHODS: A retrospective review of all consecutive, open, live-related renal transplants with double RA with significant luminal discrepancy performed at our center from January 2014 to September 2018 was undertaken. Significant luminal discrepancy was defined as smaller RA constituting 30% ± 5% of total RA diameter on preoperative computed tomography angiogram. Three groups were defined: Group A - pantaloon anastomosis, Group B - end-to-side anastomosis of smaller to main RA, and Group C - separate implantation of each artery. The primary objective was to study feasibility and safety of pantaloon anastomosis measured by recipient serum creatinine levels, Doppler ultrasound, and vascular complications (vascular thrombosis and anastomotic bleed). Secondary objectives included measurement of cold ischemia time, warm ischemia time in recipient (WIR), and nonvascular recipient complications. RESULTS: Fifty-eight recipients had donors with double RA with significant luminal discrepancy. Group A - included 40, Group B - 5, and Group C - 13 patients. Recipient creatinine at day-7, - 30, and - 90 were similar among the groups. The 30-day perioperative complication rate was also similar. Group A and B had significantly lower WIR and higher cold ischemia time compared to Group C. CONCLUSION: Pantaloon anastomosis is feasible in renal grafts with double RA with significant luminal discrepancy and offers advantage of lower WIR compared to separate implantation technique.

Type 8 long QT syndrome: pathogenic variants in CACNA1C-encoded Cav1.2 cluster in STAC protein binding site.

AIMS: Pathogenic gain-of-function variants in CACAN1C cause type-8 long QT syndrome (LQT8). We sought to describe the electrocardiographic features in LQT8 and utilize molecular modelling to gain mechanistic insights into its genetic culprits. METHODS AND RESULTS: Rare variants in CACNA1C were identified from genetic testing laboratories. Treating physicians provided clinical information. Variant pathogenicity was independently assessed according to recent guidelines. Pathogenic (P) and likely pathogenic (LP) variants were mapped onto a 3D modelled structure of the Cav1.2 protein. Nine P/LP variants, identified in 23 patients from 19 families with non-syndromic LQTS were identified. Six variants, found in 79% of families, clustered to a 4-residue section in the cytosolic II-III loop region which forms a region capable of binding STAC SH3 domains. Therefore, variants may affect binding of SH3-domain containing proteins. Arrhythmic events occurred in similar proportions of patients with II-III loop variants and with other P/LP variants (53% vs. 48%, P = 0.41) despite shorter QTc intervals (477 ± 31 ms vs. 515 ± 37 ms, P = 0.03). A history of sudden death was reported only in families with II-III loop variants (60% vs. 0%, P = 0.03). The predominant T-wave morphology was a late peaking T wave with a steep descending limb. Exercise testing demonstrated QTc prolongation on standing and at 4 min recovery after exercise. CONCLUSION: The majority of P/LP variants in patients with CACNA1C-mediated LQT8 cluster in an SH3-binding domain of the cytosolic II-III loop. This represents a 'mutation hotspot' in LQT8. A late-peaking T wave with a steep descending limb and QT prolongation on exercise are commonly seen.

Allosteric regulation of rhomboid intramembrane proteolysis.

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases.

Papillary renal cell carcinoma with abscess formation: A report of three cases.

We report three cases of renal cell carcinoma (RCC) associated with abscess formation. Such association has been reported uncommonly in literature. Our cases were unique in that final histopathological report was papillary RCC in all of the patients.

Perioperative outcomes of minimally invasive versus open radical cystectomy: A single-center experience.

INTRODUCTION: Open radical cystectomy (RC) is associated with significant morbidity and the role of minimally invasive surgery (MIS) in reducing morbidity of RC is controversial A direct comparison of various surgical modalities on perioperative outcomes is lacking in the Indian literature. We evaluated outcomes of minimally invasive (robotic and laparoscopic) versus open RC with pelvic lymph node dissection (PLND) performed at our institute from 2014 to 2016. METHODS: Eighty-three patients of RC with PLND were prospectively analyzed from December 2014 to February 2016. All patients of muscle invasive urothelial cancer of the bladder undergoing RC (open or MIS) were included in the study. Based on patients preference they were assigned to one of the three groups (Open RC, robot-assisted RC, or laparoscopic RC). Their demographic profile, preoperative disease stage, operative data like operative time, blood loss, intraoperative complications, histopathological data like pathological stage, lymph-node yield etc., postoperative complications if any and total duration of stay were recorded. These data of laparoscopic, open, and robotic cystectomies were compared in terms of various demographic, histopathologic parameters and perioperative outcomes. RESULTS: Twenty-nine patients (34.93%) underwent minimally invasive RC with PLND (5 laparoscopic and 24 robotic). The median age of patients was 58 years. Mean number of lymph nodes removed was 22.5 ± 14.6. The total number of lymph nodes removed in laparoscopic surgery was 104 with a yield of 20.6 per patient, in robotic surgery were 627 with a yield of 26.1 per patient, and in open surgery were 1119 with a yield of 20.7 per patient (P = 0.004). Clavien-Dindo Grade 2 and 3 complications were seen in 37.5% of robotic, 60% of laparoscopic, and 55.54% of open RC. Average blood loss and operative time in laparoscopic, robotic, and open RC were 511.53 ± 311.02 ml, 552.08 ± 267.63 ml, and 512.05 ± 213.9 ml and 8.23 ± 1.36 h (hrs), 7.53 ± 1.92 h, and 5.85 ± 1.76 h, respectively (P = 0.68 and <0.001, respectively). CONCLUSIONS: MIS is associated with significantly longer operative time than open RC. Robotic RC has significantly higher lymph node yield than open or laparoscopic RC. Minimally invasive RC is equivalent to open surgery in terms of perioperative morbidity, mortality, and blood loss.

Reversible Unfolding of Rhomboid Intramembrane Proteases.

Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding.

Critical Roles of Two Hydrophobic Residues within Human Glucose Transporter 9 (hSLC2A9) in Substrate Selectivity and Urate Transport.

High blood urate levels (hyperuricemia) have been found to be a significant risk factor for cardiovascular diseases and inflammatory arthritis, such as hypertension and gout. Human glucose transporter 9 (hSLC2A9) is an essential protein that mainly regulates urate/hexose homeostasis in human kidney and liver. hSLC2A9 is a high affinity-low capacity hexose transporter and a high capacity urate transporter. Our previous studies identified a single hydrophobic residue in trans-membrane domain 7 of class II glucose transporters as a determinant of fructose transport. A mutation of isoleucine 335 to valine (I355V) in hSLC2A9 can reduce fructose transport while not affecting glucose fluxes. This current study demonstrates that the I335V mutant transports urate similarly to the wild type hSLC2A9; however, Ile-335 is necessary for urate/fructose trans-acceleration exchange to occur. Furthermore, Trp-110 is a critical site for urate transport. Two structural models of the class II glucose transporters, hSLC2A9 and hSLC2A5, based on the crystal structure of hSLC2A1 (GLUT1), reveal that Ile-335 (or the homologous Ile-296 in hSLC2A5) is a key component for protein conformational changes when the protein translocates substrates. The hSLC2A9 model also predicted that Trp-110 is a crucial site that could directly interact with urate during transport. Together, these studies confirm that hSLC2A9 transports both urate and fructose, but it interacts with them in different ways. Therefore, this study advances our understanding of how hSLC2A9 mediates urate and fructose transport, providing further information for developing pharmacological agents to treat hyperuricemia and related diseases, such as gout, hypertension, and diabetes.

Successful management of renal mucormycosis with antifungal therapy and drainage.

We report a case of isolated extensive renal mucormycosis in an immunocompetent adult, who was successfully managed conservatively without surgical debridement. To the best of our knowledge, this is the first case where antifungal therapy alone was sufficient even with such an extensive involvement.

Contamination from an affinity column: an encounter with a new villain in the world of membrane-protein crystallization.

Attempts to crystallize AtNTT1, a chloroplast ATP/ADP transporter from Arabidopsis thaliana, revealed an unexpected contaminant, Strep-Tactin, a variant of streptavidin that was used during purification of the protein. Although it was present in very small amounts, crystals of Strep-Tactin were reproducibly grown from the AtNTT1 solution. AtNTT1 was overexpressed in Escherichia coli and purified from detergent-solubilized membrane fractions using Strep-Tactin affinity chromatography based on an engineered streptavidin. The contamination of protein solutions purified on Strep-Tactin columns has never been described previously and seems to be specific to membrane proteins solubilized in detergents. Trace amounts of Strep-Tactin were observed to be eluted from a Strep-Tactin column using several routinely used detergents, illustrating their possible role in the contamination. This finding raises an alarm and suggests caution in membrane-protein purification using Strep-Tactin affinity columns, where detergents are essential components. The small crystals of contaminant protein led to the structure at 1.9 Å resolution of Strep-Tactin in complex with desthiobiotin.

Clinicopathologic profile of benign breast conditions in Indian women: prospective study based on aberrations of normal development and involution classification.

BACKGROUND: Benign conditions of the breast are common, but a great deal of confusion exists in their nomenclature, classification, and, treatment protocols. The Aberration of Normal Development and Involution (ANDI) classification provides a comprehensive framework within which to correlate clinical presentation with pathogenesis. Most clinical studies have not used this classification system. We studied the spectrum of benign breast conditions in Indian patients with reference to the ANDI system and performed a clinicopathologic correlation. METHODS: This prospective study was carried out on 262 consecutive patients attending the surgery department on an outpatient basis for benign breast conditions. RESULTS: A total of 199 (76 %) patients had a diagnosis of benign breast disorder, and 44 (17 %) had benign breast disease. There were 19 (7 %) women with non-ANDI and other conditions. The spectrum of benign breast disorders included fibroadenoma 77, mastalgia with nodularity 98, galactocele 7, cysts 4, and nipple discharge (ND) 13. Benign breast diseases included giant/multiple fibroadenomas 18, incapacitating mastalgia and nodularity 13, subareolar abscess with mammary fistula 7, periductal mastitis with suppuration 4, and ND 2. Cytopathologic correlation performed in 115 patients showed nonproliferative disease in 82, proliferative disease without atypia in 19, proliferative disease with atypia in 3, and miscellaneous 11. CONCLUSIONS: The ANDI classification is a comprehensive system that delineates the borderline between normal and abnormal, achieves correlation of clinical signs and symptoms with histologic changes, and provides risk stratification and pragmatic definition of management protocols.

Biopolymeric Nanocarriers for Nutrient Delivery and Crop Biofortification.

Driven by the possibility of precise transformational change in nutrient-enrichment technology to meet global food demand, advanced nutrient delivery strategies have emerged to pave the path toward success for nutrient enrichment in edible parts of crops through bioderived nanocarriers with increased productivity. Slow and controlled release of nutrient carrier materials influences the nutrient delivery rate in soil and in the edible parts of crops with a sluggish nutrient delivery to enhance their availability in roots by minimizing nutrient loss. With a limited understanding of the nutrient delivery mechanism in soil and the edible parts of crops, it is envisaged to introduce nutrient-enrichment technology for nutrient delivery that minimizes environmental impact due to its biodegradable nature. This article attempts to analyze the possible role of the cellulose matrix for nutrient release and the role of cellulose nanocomposites and nanofibers. We have proposed a few cellulose derived biofortificant materials as nutrient carriers, such as (1) nanofibers, (2) polymer-nanocellulose-clay composites, (3) silk-fibroin derived nanocarriers, and (4) carboxymethyl cellulose. An effort is undertaken to describe the research need by linking a biopolymer derived nanocarrier for crop growth regulation and experimental nitrogen release analysis. We have finally provided a perspective on cellulose nanofibers (CNFs) for microcage based nutrient loading ability. This article aims to explain why biopolymer derived nutrient carriers are the alternative candidate for alleviating nutrient deficiency challenges which are involved in focusing the nutrient delivery profile of biopolymers and promising biofortification of crops.

The role of phosphorylation in atrial fibrillation: a focus on mass spectrometry approaches.

Atrial fibrillation (AF) is the most common arrhythmia worldwide. It is associated with significant increases in morbidity in the form of stroke and heart failure, and a doubling in all-cause mortality. The pathophysiology of AF is incompletely understood, and this has contributed to a lack of effective treatments and disease-modifying therapies. An important cellular process that may explain how risk factors give rise to AF includes post-translational modification of proteins. As the most commonly occurring post-translational modification, protein phosphorylation is especially relevant. Although many methods exist for studying protein phosphorylation, a common and highly resolute technique is mass spectrometry (MS). This review will discuss recent evidence surrounding the role of protein phosphorylation in the pathogenesis of AF. MS-based technology to study phosphorylation and uses of MS in other areas of medicine such as oncology will also be presented. Based on these data, future goals and experiments will be outlined that utilize MS technology to better understand the role of phosphorylation in AF and elucidate its role in AF pathophysiology. This may ultimately allow for the development of more effective AF therapies.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19. In Brief: LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated. Highlights: LY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variantsNo loss of potency against currently circulating variantsBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID databaseBreadth of neutralizing activity and potency supports clinical development.

Implication of Wood-Derived Hierarchical Carbon Nanotubes for Micronutrient Delivery and Crop Biofortification.

A similarity of metal alloy encapsulation with the micronutrient loading in carbon nanoarchitecture can be fueled by exploring carbon nanocarriers to load micronutrient and controlled delivery for crop biofortification. A wood-derived nanoarchitecture model contains a few-graphene-layer that holds infiltrated alloy nanoparticles. Such wood-driven carbonized framework materials with legions of open porous architectures and minimized-tortuosity units further decorated carbon nanotubes (CNTs), which originate from heat treatment to carbonized wood samples. These wood-derived samples can alleviate micronutrient nanoparticle permeation and delivery to the soil. A rapid heat shock treatment can help in distributing N-C-NiFe metal alloy encapsulation in carbon frameworks uniformly in that case; higher heating and rapid extinction of heat shock have led to formation of good dispersion of nanoparticles. The wood-carbon framework decorated with metal alloys displays promising electrocatalytic features and cyclic stability for hydrogen evolution. Envisaged from this strategy, we obtain enough evidence to form an opinion that a singular heat shock process can even lead to a strategy of faster growth of a wood-carbon network with well-dispersed micronutrient metal salts in porous matrices for high-efficiency delivery to the soil. Having envisaged the formation of ultrafine nanoparticles with a good dispersion profile in the case of transition metals and alloy encapsulation in the carbon network due to the rapid heating and quenching rates, we anticipate that the loading of micronutrients in the wood-derived nanoarchitecture of carbonized wood derived carbon nanotube (CW-CNT), which can offer an application in seed germination and enhance growth rates of crops. The experience of controlled experiments on germination of tomato seeds on a medium containing CW-CNT that can diffuse the seed coat with the promotion of water uptake inside seeds for enhanced germination and growth of tomato seedlings can be further extended to cereal crops.

Cryo-EM structures of the ABCA4 importer reveal mechanisms underlying substrate binding and Stargardt disease.

ABCA4 is an ATP-binding cassette (ABC) transporter that flips N-retinylidene-phosphatidylethanolamine (N-Ret-PE) from the lumen to the cytoplasmic leaflet of photoreceptor membranes. Loss-of-function mutations cause Stargardt disease (STGD1), a macular dystrophy associated with severe vision loss. To define the mechanisms underlying substrate binding and STGD1, we determine the cryo-EM structure of ABCA4 in its substrate-free and bound states. The two structures are similar and delineate an elongated protein with the two transmembrane domains (TMD) forming an outward facing conformation, extended and twisted exocytoplasmic domains (ECD), and closely opposed nucleotide binding domains. N-Ret-PE is wedged between the two TMDs and a loop from ECD1 within the lumen leaflet consistent with a lateral access mechanism and is stabilized through hydrophobic and ionic interactions with residues from the TMDs and ECDs. Our studies provide a framework for further elucidating the molecular mechanism associated with lipid transport and disease and developing promising disease interventions.

Primary Bladder Neck Obstruction in a Young Woman: A Novel Technique of Bladder Neck Incision Using Holmium Laser.

Background: Management of primary bladder neck obstruction (PBNO) in women is difficult. Surgical treatment in the form of bladder neck incision (BNI) is indicated in many cases. There is no defined consensus on the best surgical technique of BNI in women. We present a novel technique of BNI in such cases using pediatric cystoscope and holmium laser, which is a promising alternative to the existing management techniques. Case Presentation: A 28-year-old lady complained of obstructive lower urinary tract symptoms. Serum biochemistry revealed azotemia. Further investigation utilizing a micturating cystourethrogram showed a closed bladder neck, and urodynamic studies revealed a high bladder outlet obstruction index suggestive of PBNO. BNI was effectively performed using pediatric cystoscope and holmium laser at 3, 9, and 12 o'clock positions. Postoperatively the patient had improving renal function and clinical improvement. Conclusion: Use of pediatric cystoscope with holmium laser for BNI in females is a novel technique that offers the advantage of higher maneuverability in the female urethra, precise incision with protection of external sphincter from thermal damage, and avoidance of the complication of vesicovaginal fistula caused by posterior incisions.