Structural constraints link differences in neutralization potency of human anti-Eastern equine encephalitis virus monoclonal antibodies.

Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC50) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC50 values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope-paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses.

Surya Namaskar: As an Alternative for Aerobic Fitness.

Context: "Surya Namaskar" (SN) may be used as a need-based short-duration aerobic activity in a confined space to establish as a substitute of an equivalent routine physical training in challenging stressful conditions. Materials and Methods: Noninvasive oxygen-kinetics metabolic responses between SN and endurance work on bicycle ergometry (BE) were compared across different phases of maximal oxygen uptake percentage (%VO2 max). SN, comprising three complete rounds per min (36 beats/min of a metronome; SN consists of 12 poses per round), was performed rhythmically and continuously for 5 min to simulate an incremental BE test (25 watts/2 min at 60 rpm). Results: SN results in a significant (P < 0.05) greater increase of arteriovenous oxygen difference at 71%-80% VO2 max while keeping a low respiratory exchange ratio (P < 0.01 and 0.001) at 41%-80% VO2 max exercising state. Conclusions: SN could be an ideal form of aerobic exercise instead of BE.

Appreciation of Pattern in Diagnosis of Lung Adenocarcinoma from Cytology Specimen: Our Experience with Fine Needle Aspiration Cytology and Cell Block in a Resource Constraint Setup.

BACKGROUND AND AIMS: Advent of personalised treatment needs correct diagnosis of lung adenocarcinoma with its molecular subtyping. Minimal use of special stain or immunohistochemistry (IHC) in small specimens save material for molecular testing. Various histologic patterns in adenocarcinoma (ADC) subtypes have different prognostic implications and current recommendation is to describe these patterns in small specimens. Aim of this study was to diagnose adenocarcinoma from cytology specimens depending on adenocarcinoma pattern on fine needle aspiration smears and cell blocks. We also studied the additional role of cell blocks as a platform for special stain and IHC. MATERIALS AND METHODS: Conventional smears and cell block (CB) preparation were examined from transthoracic CT guided FNA samples of suspicious lung malignancy cases. Clear defining architectural pattern and cytomorphological features in favour of adenocarcinoma were evaluated and mucin stain and IHC were used as and when required. RESULTS: A total of 86 cases were included in this study, of which 83 cases were diagnosed as adenocarcinoma, 52 (62.5%) showed clear cut evidence of adenocarcinoma from smears and CBs. CB morphology alone aided the diagnosis in 12. Various ADC patterns in combination or alone were appreciated in these 64 cases. Sixteen needed mucin stain and 3 needed IHC for diagnosis. Forty one were ADC with solid pattern of which 39 showed high nuclear grade. CONCLUSION: Adequately cellular FNA smears and corresponding cell blocks of optimal quality can aid effectively in diagnosing adenocarcinoma and appreciating its pattern. Therefore, it would minimize the need for special stain and/or IHC with preservation of more material for molecular testing.

Tumor Cell Representation by an Improvised Technique of Fine-Needle Aspiration Specimen Acquisition and Cell Block Preparation: Our Experience in Lung Cancer Cases in a Peripheral Center of Eastern India.

BACKGROUND: Being a minimally invasive diagnostic technique, Fine-Needle Aspiration Cytology (FNAC) has become the first-line test and corresponding aspirated material has become the target specimen for diagnosis and ancillary tests in lung carcinoma. Although the role of Cell Blocks (CBs) in diagnosis and in ancillary testing is well recognized in literature, limited attention has been paid to specimen procurement and triage in the preparation of CBs. In the present scenario, CBs are not consistently optimal because of its low cellularity. AIMS: This study is aimed to describe an improvised technique of specimen acquisition and cell block preparation in CT-guided FNACs of lung carcinoma cases in a resource-constrained center and to assess its efficacy for optimal representation of cellularity, morphology, and architecture. MATERIALS AND METHODS: Total 85 lung carcinoma cases undergoing CT-guided FNAC in our center from February 2017 to January 2018 were included in this study. 4 to 5 direct smears and subsequent CBs were made from material obtained by single pass. Cellularity of smears and corresponding cell blocks were assessed and categorized according to a scoring system (score 1 to 3 for number of cells <50, 50-100, >100, respectively). Preserved architecture and morphology were also assessed in smears and CBs. RESULTS: The evaluated samples showed a cellularity score 3 in 65.4%CBs and score 2 in 24.7% CBs. Overall, 90.1% cell blocks had acceptable cellularity. Cell morphology was preserved in all CBs of acceptable cellularity, except for two adenocarcinoma, one squamous cell carcinoma, and one small cell carcinoma blocks. Cellular architecture was also preserved in all CBs of acceptable cellularity. CONCLUSIONS: This simple improvised technique of CB preparation optimized its cellularity, morphology, and architectural preservation, even after adequate cellular FNA smears.

A Novel Polar Core and Weakly Fixed C-Tail in Squid Arrestin Provide New Insight into Interaction with Rhodopsin.

Photoreceptors of the squid Loligo pealei contain a G-protein-coupled receptor (GPCR) signaling system that activates phospholipase C in response to light. Analogous to the mammalian visual system, signaling of the photoactivated GPCR rhodopsin is terminated by binding of squid arrestin (sArr). sArr forms a light-dependent, high-affinity complex with squid rhodopsin, which does not require prior receptor phosphorylation for interaction. This is at odds with classical mammalian GPCR desensitization where an agonist-bound phosphorylated receptor is needed to break stabilizing constraints within arrestins, the so-called "three-element interaction" and "polar core" network, before a stable receptor-arrestin complex can be established. Biophysical and mass spectrometric analysis of the squid rhodopsin-arrestin complex indicates that in contrast to mammalian arrestins, the sArr C-tail is not involved in a stable three-element interaction. We determined the crystal structure of C-terminally truncated sArr that adopts a basal conformation common to arrestins and is stabilized by a series of weak but novel polar core interactions. Unlike mammalian arrestin-1, deletion of the sArr C-tail does not influence kinetic properties of complex formation of sArr with the receptor. Hydrogen-deuterium exchange studies revealed the footprint of the light-activated rhodopsin on sArr. Furthermore, double electron-electron resonance spectroscopy experiments provide evidence that receptor-bound sArr adopts a conformation different from the one known for arrestin-1 and molecular dynamics simulations reveal the residues that account for the weak three-element interaction. Insights gleaned from studying this system add to our general understanding of GPCR-arrestin interaction.

Structural Basis of TPR-Mediated Oligomerization and Activation of Oncogenic Fusion Kinases.

The nuclear pore complex subunit TPR is found in at least five different oncogenic fusion kinases, including TPR-MET, yet how TPR fusions promote activation of kinases and their oncogenic activities remains poorly understood. Here we report the crystal structure of TPR(2-142), the MET fusion partner of oncogenic TPR-MET. TPR(2-142) contains a continuous 124-residue α helix that forms an antiparallel tetramer from two leucine zipper-containing parallel coiled coils. Remarkably, single mutations cause strikingly different conformations of the coiled coil, indicating its highly dynamic nature. We further show that fusion of TPR(2-142) to the MET intracellular domain strongly and selectively stabilizes the αG helix of the MET kinase domain, and mutations of only the TPR leucine zipper residues at the junction to MET, but not other leucine zipper residues, abolish kinase activation. Together, these results provide critical insight into the TPR structure and its ability to induce dimerization and activation of fusion kinases.

Tuberculosis of Distal Radius Presenting as Cystic Lesion in a Nine-Month-Old Infant: A Rare Case Report.

Tuberculous osteomyelitis affecting long bones is a rare form of tuberculosis. Among infants born in endemic region, often it is difficult to diagnose owing to its subtle clinical features. Here, a case of tuberculous osteomyelitis affecting the distal radial metaphysis is reported, presenting as a cystic lesion in a nine-month-old male infant. Open biopsy with curettage was performed followed by filling of the cavity with synthetic bone substitute (beta tricalcium phosphate granules). The diagnosis was confirmed by histopathological examination and demonstration of acid fast bacilli. Further treatment with anti-tubercular drugs led to clinical and radiological improvement. This case report highlights the importance of keeping tuberculosis as a differential diagnosis while dealing with cases with similar presentation in an endemic region.

Renal Tuberculosis Presenting as a Mass Lesion in a Two-year-old Girl: Report of a rare case.

Genitourinary tuberculosis usually occurs in young adults and the middle-aged and is very uncommon in the paediatric population. It generally presents with haematuria, pyuria, irritative voiding symptoms and flank pain; presentation as a renal mass is highly unusual. We report a two-year-old girl who was referred to the Nil Ratan Sircar Medical College, Kolkata, India, in June 2014 with abdominal pain. Subsequent radiological investigations revealed a left renal hypoechoic mass lesion. A left nephroureterectomy was performed on suspicion of a Wilms' tumour. Histopathology indicated an epithelioid granuloma with lymphocytic infiltration, suggestive of a tubercular aetiology. A Mantoux tuberculin skin test was positive; however, there was no evidence of tuberculosis detected elsewhere in the body and the source of the infection could not be identified. A diagnosis of renal tuberculosis was made and the child was treated with antitubercular drugs. The patient was asymptomatic at a six-month follow-up.

The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation.

Giant Cell Tumour of Soft Tissue in Neck: An Uncommon Tumour in an Uncommon Location.

Giant cell tumour of soft tissue is an extremely rare tumour. It is thought to be the soft tissue counterpart of giant cell tumours of the bone due to its histological and immunohistochemical resemblances. Almost 80% of these tumours occur in upper and lower extremities; neck is a very rare location. Here we describe a case of primary soft tissue giant cell tumour in right submandibular region. A 35-year-old male patient presented with a swelling in right submandibular area. FNAC suggested a benign soft tissue neoplasm, comprising of spindle cells and multinucleated giant cells. Histopathology of resected specimen showed spindle cell tumour with intricately mixed giant cells, consistent with a primary giant cell tumour of soft tissue. Giant cells were CD 68 positive. Giant cell tumour of soft tissue is benign tumour, though very rarely can show features of malignancy. We present this case for its rarity and morphological overlap with other soft tissue neoplasms containing giant cells.

Probing the role of the C-H...O hydrogen bond stabilized polypeptide chain reversal at the C-terminus of designed peptide helices. Structural characterization of three decapeptides.

The structural characterization in crystals of three designed decapeptides containing a double d-segment at the C-terminus is described. The crystal structures of the peptides Boc-Leu-Aib-Val-Xxx-Leu-Aib-Val-(D)Ala-(D)Leu-Aib-OMe, (Xxx = Gly 2, (D)Ala 3, Aib 4) have been determined and compared with those reported earlier for peptide 1 (Xxx = Ala) and the all l analogue Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe, which yielded a perfect right-handed alpha-helical structure. Peptides 1 and 2 reveal a right-handed helical segment spanning residues 1 to 7, ending in a Schellman motif with (D)Ala(8) functioning as the terminating residue. Polypeptide chain reversal occurs at residue 9, a novel feature that appears to be the consequence of a C-H.O hydrogen bond between residue 4 C(alpha)H and residue 9 CO groups. The structures of peptides 3 and 4, which lack the pro R hydrogen at the C(alpha) atom of residue 4, are dramatically different. Peptide 3 adopts a right-handed helical conformation over the 1 to 7 segment. Residues 8 and 9 adopt alpha(L) conformations forming a C-terminus type I' beta-turn, corresponding to an incipient left-handed twist of the polypeptide chain. In peptide 4, helix termination occurs at Aib(6), with residues 6 to 9 forming a left-handed helix, resulting in a structure that accommodates direct fusion of two helical segments of opposite twist. Peptides 3 and 4 provide examples of chiral residues occurring in the less favored sense of helical twist; (D)Ala(4) in peptide 3 adopts an alpha(R) conformation, while (L)Val(7) in 4 adopts an alpha(L) conformation. The structural comparison of the decapeptides reported here provides evidence for the role of specific C-H.O hydrogen bonds in stabilizing chain reversals at helix termini, which may be relevant in aligning contiguous helical and strand segments in polypeptide structures.

Functional map and domain structure of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor/scatter factor.

Little is known about the large ectodomain of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor/scatter factor (HGF/SF). Here, we establish by deletion mutagenesis that the HGF/SF and heparin-binding sites of MET are contained within a large N-terminal domain spanning the alpha-chain (amino acids 25-307) and the first 212 amino acids of the beta-chain (amino acids 308-519). Neither the cystine-rich domain (amino acids 520-561) nor the C-terminal half of MET (amino acids 562-932) bind HGF/SF or heparin directly. The MET ectodomain, which behaves as a rod-shaped monomer with a large Stokes radius in solution, binds HGF/SF in the absence or presence of heparin, and forms a stable HGF/SF-heparin-MET complex with 1:1:1 stoichiometry. We also show that the ligand-binding domain adopts a beta-propeller fold, which is similar to the N-terminal domain of alphaV integrin, and that the C-terminal half contains four Ig domains (amino acids 563-654, 657-738, 742-836, and 839-924) of the unusual structural E set, which could be modeled on bacterial enzymes. Our studies provide 3D models and a functional map of the MET ectodomain. They have broad implications for structure-function of the MET receptor and the related semaphorin and plexin proteins.

Protection by rapid chemical preconditioning of stressed hippocampal slice: a study of cellular swelling using optical scatter imaging.

It has been demonstrated that anoxic preconditioning protects against a subsequent 'lethal' injury in the hippocampal slice. The goal of this paper was to test the hypothesis that chemical preconditioning could help reduce the cellular swelling observed in excitotoxically injured hippocampal slices. The control slice was given a 10-min insult of 100 microM N-methyl-D-aspartate (NMDA) to simulate ischemic injury, followed by 30-min perfusion of standard Ringers solution. Cellular swelling was observed with a microscope designed to image light scatter changes resulting from cellular swelling. After the control NMDA injury, the average peak scatter change for CA1, CA3 and DG regions was 31.0 +/- 3.4, 22.4 +/- 4.8 and 27.6 +/- 4.6%, respectively. The peak scatter change of the overall slice was 26.0 +/- 3.6%. The experimental slices were preconditioned by three short 100 microM NMDA insults of 15 s each separated by 10 min of standard Ringers solution perfusion. The slices then received 10 min of 'lethal' injury by 100 microM NMDA. It was observed that the overall scatter signal, as a measure of cellular swelling, was reduced by 8.0% (P<0.05, n=11) after preconditioning. A regional heterogeneity in the responses was also observed. Cellular swelling in CA1, CA3 and DG were reduced by 9.8% (P<0.001, n=11), 9.2% (P<0.005, n=11) and 7.7% (P<0.05, n=11), respectively, when compared to the control. This study presents experimental evidence that short episodes of preconditioning may protect against acute cellular swelling under ischemic conditions.