Structure-guided engineering of biased-agonism in the human niacin receptor via single amino acid substitution.

The Hydroxycarboxylic acid receptor 2 (HCA2), also known as the niacin receptor or GPR109A, is a prototypical GPCR that plays a central role in the inhibition of lipolytic and atherogenic activities. Its activation also results in vasodilation that is linked to the side-effect of flushing associated with dyslipidemia drugs such as niacin. GPR109A continues to be a target for developing potential therapeutics in dyslipidemia with minimized flushing response. Here, we present cryo-EM structures of the GPR109A in complex with dyslipidemia drugs, niacin or acipimox, non-flushing agonists, MK6892 or GSK256073, and recently approved psoriasis drug, monomethyl fumarate (MMF). These structures elucidate the binding mechanism of agonists, molecular basis of receptor activation, and insights into biased signaling elicited by some of the agonists. The structural framework also allows us to engineer receptor mutants that exhibit G-protein signaling bias, and therefore, our study may help in structure-guided drug discovery efforts targeting this receptor.

Molecular insights into atypical modes of ß-arrestin interaction with seven transmembrane receptors.

ß-arrestins (ßarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of ßarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the ßarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of ßarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of ßarr2 from a ß strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-ßarr complexes with direct implications for exploring novel therapeutic avenues.

Development of a New Knee Arthroscopy Operative Proforma Saving Thousands of British Pounds.

Coding inaccuracies in documentation of surgical procedures misrepresent the productivity of departments, with harmful fiscal consequences and detract from effective clinical governance. We aimed to assess the extent of this within our centres.  We retrospectively analysed the operative records of 34 patients from two centres over a period of a month, undergoing varying arthroscopic knee operations. We found that 50% of cases had incorrect coding for procedures performed. On review of the clinical coding, the loss of payment summed up to £29,325.  The flawed coding practices stemmed from the heterogeneity and convolution in documentation of procedures. Our intervention was the development of a multi-faceted arthroscopic operation note proforma, centred on concise documentation for appropriate codes to be gleaned. We re-audited our new proforma, retrospectively collating data on 37 patients over a period of five months undergoing arthroscopic knee procedures. We found only 5% of cases were coded incorrectly, summing to a loss in tariff payment of £2654.  In conclusion, poor quality of documentation and written communication between surgical and coding departments can have drastic ramifications for funding. An active refinement of this process can ultimately help to provide more resources for improved patient care.

Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors.

The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.

Wound healing strategies based on nanoparticles incorporated in hydrogel wound patches.

The intricate, tightly controlled mechanism of wound healing that is a vital physiological mechanism is essential to maintaining the skin's natural barrier function. Numerous studies have focused on wound healing as it is a massive burden on the healthcare system. Wound repair is a complicated process with various cell types and microenvironment conditions. In wound healing studies, novel therapeutic approaches have been proposed to deliver an effective treatment. Nanoparticle-based materials are preferred due to their antibacterial activity, biocompatibility, and increased mechanical strength in wound healing. They can be divided into six main groups: metal NPs, ceramic NPs, polymer NPs, self-assembled NPs, composite NPs, and nanoparticle-loaded hydrogels. Each group shows several advantages and disadvantages, and which material will be used depends on the type, depth, and area of the wound. Better wound care/healing techniques are now possible, thanks to the development of wound healing strategies based on these materials, which mimic the extracellular matrix (ECM) microenvironment of the wound. Bearing this in mind, here we reviewed current studies on which NPs have been used in wound healing and how this strategy has become a key biotechnological procedure to treat skin infections and wounds.

Structural snapshots uncover a key phosphorylation motif in GPCRs driving ß-arrestin activation.

Agonist-induced GPCR phosphorylation is a key determinant for the binding and activation of ß-arrestins (ßarrs). However, it is not entirely clear how different GPCRs harboring divergent phosphorylation patterns impart converging active conformation on ßarrs leading to broadly conserved functional responses such as desensitization, endocytosis, and signaling. Here, we present multiple cryo-EM structures of activated ßarrs in complex with distinct phosphorylation patterns derived from the carboxyl terminus of different GPCRs. These structures help identify a P-X-P-P type phosphorylation motif in GPCRs that interacts with a spatially organized K-K-R-R-K-K sequence in the N-domain of ßarrs. Sequence analysis of the human GPCRome reveals the presence of this phosphorylation pattern in a large number of receptors, and its contribution in ßarr activation is demonstrated by targeted mutagenesis experiments combined with an intrabody-based conformational sensor. Taken together, our findings provide important structural insights into the ability of distinct GPCRs to activate ßarrs through a significantly conserved mechanism.

A streamlined protocol for expression and purification of wild-type ß-arrestins.

The two isoforms of ß-arrestins namely ß-arrestin 1 and 2 interact with, and regulate a broad repertoire of G protein-coupled receptors (GPCRs). While several protocols have been described in the literature for purification of ß-arrestins for biochemical and biophysical studies, some of these protocols involve multiple complicated steps that prolong the process and yield relatively smaller amounts of purified proteins. Here, we describe a simplified and streamlined protocol for expression and purification of ß-arrestins using E. coli as an expression host. This protocol is based on N-terminal fusion of GST tag and involves a two-step protocol involving GST-based affinity chromatography and size exclusion chromatography. The protocol described here yields sufficient amounts of high-quality purified ß-arrestins suitable for biochemical and structural studies.

Generating (Factual?) Narrative Summaries of RCTs: Experiments with Neural Multi-Document Summarization.

We consider the problem of automatically generating a narrative biomedical evidence summary from multiple trial reports. We evaluate modern neural models for abstractive summarization of relevant article abstracts from systematic reviews previously conducted by members of the Cochrane collaboration, using the authors conclusions section of the review abstract as our target. We enlist medical professionals to evaluate generated summaries, and we find that summarization systems yield consistently fluent and relevant synopses, but these often contain factual inaccuracies. We propose new approaches that capitalize on domain-specific models to inform summarization, e.g., by explicitly demarcating snippets of inputs that convey key findings, and emphasizing the reports of large and high-quality trials. We find that these strategies modestly improve the factual accuracy of generated summaries. Finally, we propose a new method for automatically evaluating the factuality of generated narrative evidence syntheses using models that infer the directionality of reported findings.

A prospective study on COVID-19 convalescent plasma donor (CCP) recruitment strategies in a resource constrained blood centre.

Background and Objectives: The COVID-19 pandemic has spread across 87 million people with more than 1·8 million deaths in the world. As there is no definite treatment modality, the use of convalescent plasma has become increasingly popular worldwide. This study aimed to identify an appropriate strategy of donor recruitment and to evaluate the appropriateness of pre-set plasma donation guidelines. Material and Methods: In this prospective study conducted from May to September 2020, the donors were recruited under the following two circumstances: Group I, patients in the post-COVID-19 follow-up in the clinic, and Group II, patients recovered from COVID-19 recruited through mass and electronic media. A pre-set donor selection criteria and laboratory investigation was designed according to national and international guidelines. Approximately 500 ml of COVID-19 convalescent plasma (CCP) was collected from recovered individuals in each group by two different cell separators. The overall donor's attendance rate, deferral rate, adverse events and donor compliance was analysed and compared between the two groups. Results: There was a significant difference in attendance in relation to registration between the groups (P < 0·0001). Donor deferral was significantly higher in group II compared with group I. The single most frequent cause of donor deferral was low antibody index (P = 0·0001). The total donor adverse event rate in CCP donation was significantly lower compared with routine plateletpheresis procedures. The donor's compliance to blood centre's protocol was satisfactory in both the groups. Conclusion: Recruitment of patients in the post-COVID-19 follow-up in the clinic was more effective than the general recruitment through mass and electronic media for convalescence plasma donation in a resource-constrained blood centre.

A computed tomography-based classification of Hoffa fracture: Surgical treatment considerations and prognostic outcome with assessment of reproducibility.

INTRODUCTION: Isolated fracture of femoral condyle in the coronal plane (Hoffa fracture) is rare and surgically challenging to treat. The purpose of this study is to introduce a new classification for this fracture, evaluating its utility in treatment guidelines and its reproducibility. MATERIALS AND METHODS: 103 patients with Hoffa fracture were operated in our tertiary care centre from 2004 to 2014. Computed tomography (CT) was done in all cases preoperatively. Many fractures had variable patterns which could not be defined and categorized as per Letennuer's classification. A pilot study with first 25 cases were analysed and certain morphological fracture patterns were observed in CT. The fixation methods and failure models were also noted. This resulted in development of a new classification specifying the fracture patterns and the preferred fixation methods for a better outcome. Next 78 consecutive fractures were then classified preoperatively as per our CT classification and treatment executed according to the protocol. Outcomes were measured using Neer's scoring system and International Knee Society Documentation Committee (IKDC) Functional Score. RESULTS: Out of 78 patients, 69 had excellent outcome, 7 had satisfactory and 2 had unsatisfactory outcome which corresponded with the specified fracture severity pattern. Uniformly excellent results were obtained in all simple pattern fractures, whereas comminuted fractures especially posterior cortex comminution and articular comminution yielded less favourable outcomes. CONCLUSION: We conclude that therapeutic outcome is significantly affected by articular comminution, posterior cortex comminution, achieving anatomical reduction and stable fixation. Pre-operative fracture classification and treatment guidelines gives a predictable outcome with least complications and has good interobserver reliability thus instrumental in surgical planning. LEVEL OF EVIDENCE: III.

Bronchiectasis and Focal Segmental Glomerulosclerosis in Rheumatoid Arthritis.

A 28-year-old male patient who was a nonsmoker presented with bilateral symmetrical polyarthritis and polyarthralgia, suggestive of rheumatoid arthritis (RA), along with shortness of breath, fever and cough, suggestive of chronic renal failure and nephrotic range proteinuria. The chest radiograph was suggestive of panacinar emphysematous changes with bilateral central bronchiectasis. The patient reported that two of his brothers had died in their third decade because of renal failure. Renal biopsy showed focal and segmental glomerulosclerosis (FSGS). FSGS with panacinar emphysema and bronchiectasis is a rare entity in RA patients, and considering the possibilities of a familial pattern of FSGS, transient receptor potential cation channel 6 channelopathy was the most valid diagnosis.

Dimerization of the plant molybdenum insertase Cnx1E is required for synthesis of the molybdenum cofactor.

The molybdenum cofactor (Moco) is a redox active prosthetic group, essentially required for numerous enzyme-catalyzed two electron transfer reactions. Moco is synthesized by an evolutionarily old and highly conserved multistep pathway. In the last step of Moco biosynthesis, the molybdenum center is inserted into the final Moco precursor adenylated molybdopterin (MPT-AMP). This unique and yet poorly characterized maturation reaction finally yields physiologically active Moco. In the model plant Arabidopsis, the two domain enzyme, Cnx1, is required for Moco formation. Recently, a genetic screen identified novel Arabidopsis cnx1 mutant plant lines each harboring a single amino acid exchange in the N-terminal Cnx1E domain. Biochemical characterization of the respective recombinant Cnx1E variants revealed two different amino acid exchanges (S197F and G175D) that impair Cnx1E dimerization, thus linking Cnx1E oligomerization to Cnx1 functionality. Analysis of the Cnx1E structure identified Cnx1E active site-bound molybdate and magnesium ions, which allowed to fine-map the Cnx1E MPT-AMP-binding site.

Diffuse parenchymal lung disease in a case of chronic arsenic exposure.

A 42-year-old housewife, the resident of rural part of West Bengal, presented with gradually progressive exertional dyspnea associated with a dry cough for last 3 years clinical features were suggestive of diffuse parenchymal lung disease (DPLD). Her chest X-ray posteroanterior view and high resolution computed tomography scan of the thorax showed bilateral patchy ground glass opacities and reticulonodular pattern. Search for the etiology revealed classical skin findings of chronic arsenic exposure in the form of generalized darkening and thickening of skin and keratotic lesions over the palms and soles and classical raindrop pigmentation over leg which was present for last 7 years subsequently her bronchoalveolar lavage fluid, hair, nail, and drinking water showed significant amount of arsenic contamination. By exclusion of all known causes of DPLD, we concluded that it was a case of DPLD due to chronic arsenic exposure. To the best of our knowledge, only few case report of DPLD in chronic arsenicosis has been reported till date.

Mature cystic teratoma of mediastinum with pleural effusion: An uncommon entity.

Teratomas are tumours composed of tissues originating from at least two of the three primitive germ layers, i.e., ectoderm, mesoderm and endoderm. A dermoid cyst, commonly known as benign cystic teratoma is a histological variant containing epidermis and its derivative (sebaceous material) within a lining of squamous epithelium. Mediastinum is the most common extragonadal site for germ cell tumours including mature teratomas. However, pleural effusion secondary to rupture of benign germ cell tumour is quite a rarity and the nature of pleural effusion in such ruptured cases has not been discussed in detail in literatures. Here, we are presenting a case of mature cystic teratoma coexistent with pleural effusion which is quite an uncommon entity making this case quite an interesting learning curve.

Silicosis in the form of progressive massive fibrosis: A diagnostic challenge.

Silicosis is a major occupational lung disease with a relatively fatal and irreversible outcome. Early diagnosis for shifting the potential candidates to safe modes of workplace as well as for prevention of further progression is the cornerstone of management. Here, we present a complicated case of silicosis in the form of progressive massive fibrosis, which was initially interpreted as tuberculosis; radiological images had resemblance with tuberculosis and cryptogenic organizing pneumonia. Radiology-guided trucut biopsy was imperative to confirm the diagnosis.

Mesothelioma with superior vena cava obstruction in young female following short latency of asbestos exposure.

An 18 years female was admitted with right-sided chest pain, dry cough, and low-grade fever and weight loss for last 1 month. On examination, patient had features of superior vena cava (SVC) syndrome with right-sided pleural effusion. Chest X-ray showed mediastinal widening with nonhomogenous opacity mainly in the periphery of right upper and mid zone with right-sided pleural effusion. Ultrasonography thorax confirmed mild pleural effusion. Pleural fluid analysis showed lymphocytic, exudative, low adenosine deaminase with negative for Pap smear. Contrast-enhanced computed tomography (CT) thorax revealed large extensive nodular soft tissue lesion along right mediastinum as well as costal pleura, with enlarged pretracheal lymphadenopathy and SVC obstruction. CT guided Tru-cut biopsy report came as malignant epithelial tumor with polygonal shape, abundant eosinophilic cytoplasm and nuclei with prominent nucleoli suggestive of mesothelioma of epithelioid type. The tumor cell expressed calretinin, WT-1, and immunonegative for thyroid transcription factor-1.

Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferases.

During the biosynthesis of heme d(1), the essential cofactor of cytochrome cd(1) nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-L-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-L-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a "puckered" conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.