Extra digital glomus tumor causing scapulothoracic impingement: A case report.

Glomus tumor is a painful benign neoplasm which arises from the glomus body. The glomus body is neuromyoarterial receptor responsible for regulating the peripheral skin blood flow and local temperature. The classical clinical symptoms of the glomus tumor are acute local pain, pin point tenderness and hypersensitivity to cold. The commonest location of the glomus tumor is the subungual region of the distal phalanx of the fingers and toes. We hereby describe a very rare case of extradigital glomus tumor in the interspace between the scapula and the posterior chest wall causing paroxysmal severe pain on the movement of the right shoulder. Being deep in location, the tumor was not clinically palpable or identifiable. The lesion was eventually located on MRI. Surgical resection of the tumor led to complete resolution of patient's symptoms.

Screening and Production of Recombinant Human Proteins: Ligation-Independent Cloning.

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the Structural Genomics Consortium, we opted for the ligation-independent cloning (LIC) method which provides the throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).

Screening and Production of Recombinant Human Proteins: Protein Production in E. coli.

In Chapter 3 , we described the Structural Genomics Consortium (SGC) process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or structural studies (e.g., crystallization or cryo-EM experiments). Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.

Screening and Production of Recombinant Human Proteins: Protein Production in Insect Cells.

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing, and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 mL of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures.

Expression Screening of Human Integral Membrane Proteins Using BacMam.

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the BacMam system. This eukaryotic expression system was selected and a screening process established in 2016 to enable production of highly challenging human integral membrane proteins (IMPs), which are a significant component of our target list. Here, we discuss our recently developed platform for identifying expression and monodispersity of IMPs from 3 mL of HEK293 cells.

Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1.

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.

A Patient of Tumour Induced Osteomalacia Undiagnosed for 11 years.

We report a 45 years old woman, bedridden due to severe bone pain, back pain, multiple spontaneous fractures over 10 years. She had low serum Phosphates. We detected a swelling in her right groin and suspected tumour induced osteomalacia. Resection of the tumour led to reversal of metabolic bone disease. Patient became ambulatory within 6 weeks of tumour resection.

Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2).

Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.

Synthesis and Antitubercular Activity of New Benzo[b]thiophenes.

In vitro and ex vivo efficacies of four series of benzo[b]thiophene-2-carboxylic acid derivatives were studied against Mycobacterium tuberculosis H37Ra (MTB). Benzo[b]thiophenes were also tested in vitro against multidrug resistant Mycobacterium tuberculosis H37Ra (MDR-MTB), and 7b was found to be highly active against A- and D-MDR-MTB/MTB (MIC ranges 2.73-22.86 µg/mL). The activity of all benzo[b]thiophenes against M. bovis BCG (BCG) was also assessed grown under aerobic and under conditions of oxygen depletion. Compounds 8c and 8g showed significant activity with MICs of 0.60 and 0.61 µg/mL against dormant BCG. The low cytotoxicity and high selectivity index data against human cancer cell lines, HeLa, Panc-1, and THP-1 indicate the potential importance of the development of benzo[b]thiophene-based 1,3-diketones and flavones as lead candidates to treat mycobacterial infections. Molecular docking studies into the active site of DprE1 (Decaprenylphosphoryl-ß-d-ribose-2'-epimerase) enzyme revealed a similar binding mode to native ligand in the crystal structure thereby helping to understand the ligand-protein interactions and establish a structural basis for inhibition of MTB. In summary, its good activity in in vitro and ex vivo model, as well as its activity against multidrug-resistant M. tuberculosis H37Ra in a potentially latent state, makes 7b an attractive drug candidate for the therapy of tuberculosis.

Antioxidant and antiproliferative activity of indigocarpan, a pterocarpan from Indigofera aspalathoides.

OBJECTIVES: This study aimed to investigate the anticancer potential of indigocarpan (1), a pterocarpan isolated from Indigofera aspalathoides, a plant found in India which has been used in Ayurveda for centuries for the treatment of oedematous tumours. METHODS: The antiproliferative activity in a panel of four human cancer cell lines was studied. The mechanism of its antiproliferative activity in human colorectal adenocarcinoma LS174T cells was investigated in detail. KEY FINDINGS: Indigocarpan (1) showed antiproliferative activity in a panel of four human cancer cell lines with IC50 s ranging from 180 to 250 µm. Indigocarpan induces p53-dependent p21 upregulation and apoptosis in LS174T cells, upregulates p53 and p21(WAF1) protein levels, enhances cleavage of caspase-3 and downregulates cyclin D1, cyclin B1 and PCNA protein levels, indicating its role in modulating cell cycle progression. Indigocarpan also exhibited a strong antioxidative effect in LS174T cells. CONCLUSIONS: Along with the antiproliferative capacity, the strong antioxidative property of the compound makes it a promising candidate for further development as an anticancer and chemopreventive compound.

Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities.

The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties.

Primary Liver Sinusoidal Non-Hodgkin's Lymphoma Presenting as Acute Liver Failure.

We describe a case of a middle-aged woman, who presented to us with fever, anorexia, abdominal distension from a massive hepatomegaly, low hemoglobin, and acute liver failure. A liver biopsy revealed B cell non-Hodgkin's lymphoma predominantly in the sinusoids with CD10, CD20, and Bcl-2 positive on immunohistochemistry. She initially responded well to chemotherapy but succumbed 6 months later to the recurrence of disease. Sinusoidal non-Hodgkin's lymphoma of the liver should be considered in the differential diagnosis of a patient with large hepatomegaly presenting with acute liver failure.

Sclerotium rolfsii lectin induces stronger inhibition of proliferation in human breast cancer cells than normal human mammary epithelial cells by induction of cell apoptosis.

Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galß1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent.

AML M7 Misdiagnosed as ALL.

Hematogones, which are normal precursors of B lymphocytes in the bone marrow, may be mistaken for blast cells on flow cytometry and histology when their numbers increase. We report such a case in a 16 months old male who was unsuccessfully treated for a pre-B cell ALL on the basis of flow cytometry of the bone marrow which showed a substantial population of CD19 and CD10 expressing 'blast' cells. A diagnosis of AML M7 was made on a subsequent bone marrow biopsy in which the blast cells expressed CD61 and Factor VIII, while they were negative for CD10 and CD20. Also present were a few CD10 and CD20 expressing small lymphoid cells, which were interpreted as hematogones. This report reiterates the problem of mistaking hematogones for 'blast' cells on flow cytometry, especially in the marrow of very young children where hematogones are often prominent.

Medium-throughput production of recombinant human proteins: ligation-independent cloning.

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the SGC, we opted for the Ligation-Independent Cloning (LIC) method which provides the medium throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in a 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).

Medium-throughput production of recombinant human proteins: protein production in E. coli.

In Chapter 4 we described the SGC process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or crystallization experiments. Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.

Medium-throughput production of recombinant human proteins: protein production in insect cells.

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 ml of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures. Although not discussed in this chapter, the same process can also be applied to integral membrane proteins (IMPs) with slight adaptations to the purification procedure.

Structural insights into functional overlapping and differentiation among myosin V motors.

Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contribute to understanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors.

The PHD and chromo domains regulate the ATPase activity of the human chromatin remodeler CHD4.

The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2ß) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex.

Lymphadenopathy resulting from acute toxoplasmosis mimicking relapse of non-Hodgkin's lymphoma on fluorodeoxyglucose positron emission tomography/computed tomography.

We report a case documenting fluorodeoxyglucose (FDG) accumulation in cervical, supraclavicular and axillary lymph nodes resulting from acute toxoplasmosis. A 50-year-old Indian female with history of non-Hodgkin's lymphoma (NHL) of left breast, postchemotherapy status, was found to have hypermetabolic right cervical, supraclavicular and axillary lymph nodes on a surveillance FDG positron emission tomography/computed tomography (PET/CT) scan. Her previous two PET/CT scans were unremarkable with no evidence of metabolically active disease. Therefore, a differential diagnosis of relapse of NHL versus infectious/inflammatory pathology was raised in the report. Biopsy of axillary lymph node demonstrated features characteristic of toxoplasmosis. The serological test results were also compatible with acute toxoplasmosis infection. Infective and inflammatory diseases are known to accumulate FDG, resulting in false positives for malignancy. This case demonstrates lymph nodal toxoplasmosis as a potential cause of false positive FDG PET/CT findings in patients with known malignancy and highlights the importance of histopathological and laboratory correlation for the accurate interpretation of FDG PET/CT scans.