Clinical-radiological features and diagnostic modalities for mediastinal melioidosis.

Melioidosis is a potentially life-threatening infection caused by the Gram-negative bacillus Burkholderia pseudomallei. Mediastinal melioidosis has a range of clinical presentations, making it difficult to diagnose: we therefore reviewed the evidence on the clinical characteristics, radiological features and invasive diagnostic modalities or interventions. An electronic search was conducted on three databases (PubMed, SCOPUS, Google Scholar) from November to December 2022. The initial search yielded 120 results, of which 34 studies met the inclusion criteria, but only 31 full-texts were retrievable. Among these, 4 were cohort studies, 26 case reports or series and 1 a conference abstract. The four main themes covered were mediastinal melioidosis as a diagnostic dilemma, unexpected complications, invasive interventions or an accompanying thoracic feature. Radiological manifestations included matting, necrosis and abscess-like collection. Severe presentations of mediastinal melioidosis included superior vena cava obstruction, sinus tract formation and pericardial tamponade. Transbronchial needle aspiration was the most common invasive diagnostic modality. Further research is needed to understand the relationship between the thoracic features of melioidosis on patient prognosis, its relationship to melioidosis transmission and potential preventive measures.

Pleural fluid residue as a diagnostic tool for cytology-negative malignant pleural effusion: A proof-of-concept study.

Background: Pleural fluid residue, or macroscopic tissue, circulating freely in the pleural fluid obtained through direct filtration, may carry diagnostic histopathological information. We aimed to determine the histopathological concordance of pleural fluid residue in diagnosing TPE and MPE, compared with conventional pleural biopsy. This was a prospective cohort study of consecutive inpatients with cytology-negative exudative effusion who underwent pleuroscopy and had their initial suctioned pleural fluid filtered for residue samples. Pleural fluid residue demonstrated malignant cells in four out of seven cases of pleural biopsy-confirmed malignancy. Pleural fluid residue has comparable cytomorphology but reduced cellularity compared with pleural biopsy. No tuberculous histological features were present in the pleural fluid residue samples. In this preliminary study pleural fluid residue provided histopathological information for malignant pleural effusion, but no incremental diagnostic information for tuberculous effusion. However larger and more definitive studies are required to clarify these findings, and to explore the utility and suitability of pleural fluid residue for mutational analysis. What the study adds: This study demonstrates the potential of pleural fluid residue as a non-invasive diagnostic method for confirming malignancy in cytology-negative exudative effusion. What are the implications of the findings: In resource-limited settings or patients contraindicated for pleural biopsy, pleural fluid residue may provide a viable diagnostic alternative; however, this observation needs further validation.

Individualised second line anti-tuberculous therapy for an extensively resistant pulmonary tuberculosis (XDR PTB) in East Malaysia.

Clinical experience with extensively Drug Resistant tuberculosis (XDR-TB) has not been reported in Malaysia before. We describe the clinical characteristics, risk factors, progress and therapeutic regimen for a healthcare worker with XDR-TB, who had failed therapy for multidrug resistant TB (MDR TB) in our institution. This case illustrates the risk of TB among healthcare workers in high TB-burden settings, the importance of obtaining upfront culture and susceptibility results in all new TB cases, the problem of acquired drug resistance developing during MDR-TB treatment, the challenges associated with XDR-TB treatment regimens, the value of surgical resection in refractory cases, and the major quality of life impact this disease can have on young, economically productive individuals.

A rare double pathology- coexistent large cell neuroendocrine cell carcinoma of the lung with Basal cell carcinoma of the skin.

No abstract available.

A Rare Double Pathology- Coexistent Large Cell Neuroendocrine Cell Carcinoma of the Lung with Basal Cell Carcinoma of the Skin

No abstract available.

Rapid screening for HIV-1 protease inhibitor leads through X-ray diffraction.

Knowledge of the three-dimensional structures of HIV-1 protease and of its complexes with various inhibitors has played a key role in development of drugs against AIDS. Hexagonal crystals of unliganded tethered HIV-1 protease in which the enzyme conformation is identical to its ligand-bound state can be used in combination with the soaking method in order to identify potential inhibitor leads via X-ray diffraction. The advantages of the soaking method are the generality of application and the rapidity of structure determination for iterative structure-based drug design. Structures of two ligand complexes with HIV-1 protease determined using this method are shown to be very similar to the structures obtained earlier via co-crystallization.

Effects of remote mutation on the autolysis of HIV-1 PR: X-ray and NMR investigations.

Autolysis rates of the C95M and C95M/C1095A mutants of a HIV-1 protease tethered dimer have been determined by real time NMR and it is observed that the double mutant has approximately two times higher rate. X-ray structure of the C95M/C1095A double mutant has been solved and refined to 2.1 A resolution. Comparison of the double mutant structure with that of C95M single mutant reveals that there is a shift in the position of the catalytic aspartates and the bound catalytic water. The mutation also causes a loss of hydrophobic packing near the dimerization domain of the protein. These observations demonstrate that subtle changes are adequate to cause significant changes in the rate of autolysis of the double mutant. This provides a rationale for the effects of remote mutations on the activity and drug resistance of the enzyme.

1.9 A x-ray study shows closed flap conformation in crystals of tethered HIV-1 PR.

Three-dimensional structure of an asymmetrically mutated (C95M) tethered human immunodeficiency virus type 1 protease enzyme (HIV-1 PR) has been determined in an unliganded form using X-ray diffraction data to 1.9 A resolution. The structure, refined using X-PLOR to an R factor of 19.5%, is unexpectedly similar to the ligand-bound native enzyme, rather than to the ligand-free native enzyme. In particular, the two flaps in the tethered dimer are in a closed configuration. The environments around M95 and C1095 are identical, showing no structural effect of this asymmetric mutation at position 95. Oxidation of Cys1095 has been observed for the first time. There is one well-defined water molecule that hydrogen bonds to both carboxyl groups of the essential aspartic acids in the active site. Proteins 2001;43:57-64.

Purification, crystallisation and preliminary X-ray diffraction study of ribosome inactivating protein: saporin.

We report here the crysallisation and molecular replacement results on the structure determination of S-9 isoform of the ribosome inactivating protein saporin. The protein was purified to homogeneity by a simple and efficient protocol. The crystals belong to the space group I4l with a = b = 91.47 A, c = 150.66 A and contain two molecules in the asymmetric unit.

X-ray structure of gelonin at 1.8 A resolution.

Gelonin is a single chain ribosome inactivating protein (RIP) with potential application in the treatment of cancer and AIDS. Diffraction quality crystals grown using PEG3350, belong to the space group P21, with a = 49.4 A, b = 44.9 A, c = 137.4 A and beta = 98.4 degrees, and contain two molecules in the asymmetric unit. Diffraction data collected to 1.8 A resolution has a Rm value of 7.3%. Structure of gelonin has been solved by the molecular replacement method, using ricin A chain as the search model. Crystallographic refinement using X-PLOR resulted in a model for which the r.m.s deviations from ideal bond lengths and bond angles are 0.012 A and 2.7 degrees, respectively. The final R-factor is 18.4% for 39,806 reflections for which I > 1.0 sigma (I). The C alpha atoms of the two molecules in the asymmetric unit superpose to within 0.38 A for 247 atom pairs. The overall fold of gelonin is similar to that of other RIPs such as ricin A chain and alpha-momorcharin, the r.m.s.d. for C alpha superpositions being 1.3 and 1.4 A, respectively. The catalytic residues (Glu166, Arg169 and Tyr113) in the active site form a hydrogen bond scheme similar to that observed in other RIPs. The conformation of Tyr74 in the active site, however, is significantly different from that in alpha-momorcharin. Three well defined water molecules are located in the active site cavity, and one of them, X319, superposes to within 0.2 A of a corresponding water molecule in the structure of alpha-momorcharin. Any of the three could be the substrate water molecule in the hydrolysis reaction catalysed by gelonin. Difference electron density for a N-linked sugar moiety has been observed near only one of the two potential glycosylation sites in the sequence. The amino acid at position 239 has been established as Lys by calculation of omit electron density maps. The two cysteine residues in the sequence, Cys44 and Cys50, form a disulphide bond, and are therefore not available for disulphide conjugation with antibodies. Based on the structure, the region of the molecule that is involved in intradimer interactions is suggested to be suitable for introducing a Cys residue for purposes of conjugation with an antibody to produce useful immunotoxins.

Drug-protein interactions. Refined structures of three sulfonamide drug complexes of human carbonic anhydrase I enzyme.

N-unsubstituted sulfonamide drugs are widely used for opthalmic disorders. Inhibition of carbonic anhydrase enzyme is believed to be the chief reason for their therapeutic effects. Structures of three such sulfonamide drugs complexed to human carbonic anhydrase I enzyme (HCAI) refined crystallographically at 2 A resolution are reported here. The drug molecules are all bound in the active site of the enzyme, but among themselves show differences in the orientations of the sulfamido groups interacting with the essential zinc ion in the active site. The activity linked solvent molecule coordinated to zinc in the native enzyme is displaced by all the three sulfonamides. The active site loop of Leu198, Thr199 and His200 has been identified to be important for binding of the drug molecules due to their appreciable atomic displacements and intra-molecular hydrogen bonds arising out of their interactions with the sulfonamides. These interactions along with active site charge requirements are proposed to be responsible for the orientational differences of the sulfamido groups and also for differences in the inhibitory powers of the drugs. A hydrogen bond network involving solvent molecules and active site residues His200 and His67 amongst others in the native enzyme, is disrupted upon binding of methazolamide but not in the other two sulfonamides. This is the first crystallographic evidence of the possible involvement of His200 in the inhibition of HCAI. An important role of Thr199 in distinguishing between the substrate and inhibitor binding modes of HCO3- to the enzyme at high pH is also inferred.

Differences in anionic inhibition of human carbonic anhydrase I revealed from the structures of iodide and gold cyanide inhibitor complexes.

The crystal structures of two anionic inhibitor complexes of human carbonic anhydrase I (HCAI), namely, HCAI-iodide and HCAI-Au(CN)(2)(-), have been refined by the restrained least-squares method at 2.2 and 2 A nominal resolution, respectively, with good stereochemistry for the final models. The R values have improved from 30.3 to 16.6% for HCAI-iodide and from 28.8 to 17.1% for HCAI-Au(CN)(2)(-). The sites of inhibitor binding as elucidated are totally different in the two structures. The iodide anion replaces the zinc-bound H(2)O/OH(-) ligand and renders the enzyme inactive. This result confirms that the zinc-bound H(2)O/OH(-) is the activity-linked group in carbonic anhydrase enzymes. Au(CN)(2)(-) binds at a different and new site near the zinc ion, without liganding to the metal. The N atom of Au(CN)(2)(-) is within hydrogen-bonding distance of the zinc-bound H(2)O/OH(-) group which shifts by about 0.4 A away from the zinc ion in relation to its position in the native HCAI. It is proposed that the presence of the inhibitor Au(CN)(2)(-) results in a conformational reorientation of the activity-linked group, due to hydrogen-bond formation with the inhibitor, which in turn sterically hinders the binding of the substrate CO(2) molecule in the active site, leading to the inhibition of HCAI enzyme.

Enzyme-substrate interactions. Structure of human carbonic anhydrase I complexed with bicarbonate.

The structure of HCAI-HCO3- complex has been refined with 10-1.6A X-ray diffraction data to an R-value of 17.7%. The structure reveals monodentate binding of the HCO3- anion at an apical tetrahedral position to the zinc ion. The binding mode and interactions of HCO3- in HCAI differ from that in HCAII. The activity linked H2O/OH- group in the free HCAI is replaced by the hydroxyl group of the bicarbonate anion. This result rules out the rearrangement of the bound HCO3- advocated earlier to explain the microscopic reversibility of the catalysed reaction. From the geometry of the H-bonds between Glu106-Thr199 pair and Glu117-His119 couple, the glutamic acids are expected to be ionized and accept H-bonds from their partners. The product-inhibiton by HCO3- anion is explained on the basis of proton localization on His119 in the Glu117-His119 couple. These results are consistent with the hypothesis that Glu117-His119 tunes the ionicity of the Zn2+ and the binding strength of HCO3- anion. A pi hydrogen bond is observed between a water and phenyl ring of the Tyr114 residue.

Crystallization of a scRIP-gelonin isolated from plant seeds Gelonium multiforum.

Single crystals of the protein gelonin isolated from the seeds of Gelonium multiforum have been grown at room temperature by vapor diffusion method. The crystals are monclinic with a = 49.4 A, b = 44.9 A, c = 137.4 A, and beta = 98.3 degrees. The space group is P2(1), with two molecules in the asymmetric unit which are related by a noncrystallographic 2-fold axis along psi = 13 degrees and phi = 88 degrees. The crystals diffract X-rays to high resolution, making it possible to obtain an accurate structure of this single chain ribosome inactivating protein.

Crystallization and X-ray analysis of a multienzyme complex containing RUBISCO and RuBP.

Single crystals of a multienzyme complex isolated from spinach leaves, and containing RUBISCO bound to the substrate RuBP have been grown and characterized. The crystals belong to the orthorhombic space group P2(1)2(1)2 with a = 173 A, b = 134 A and c = 112 A, and contain two enzyme complex molecules in the unit cell. Diffraction data to 2.5 A resolution have been collected on the sychrotron source at the photon factory in Japan. Initial structure determination has been carried out using the molecular replacement method. The RUBISCO molecule in the complex has the normal L8S8 subunit configuration, and difference electron density is clearly observed for the other component enzymes and the RuBP substrate.

Solution conformation of d-CTCGAGCTCGAG by two-dimensional NMR: conformational heterogeneity at XhoI cleavage site.

Nonexchangeable proton resonances in the 500-MHz NMR spectrum of d-CTCGAGCTCGAG have been assigned by using two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). 1H-1H coupling constants (J) in the deoxyribose rings have been measured by analyzing intensity and multiplet patterns in the phase-sensitive omega 1-scaled COSY spectra. A modification of the J-resolved technique, called amplitude-modulated J-resolved spectroscopy, has been described and used to increase the accuracy of J measurements. Absorption mode omega 1-scaled NOESY spectra at mixing times in the range 50-200 ms have been analyzed to monitor spin diffusion. A 50-ms spectrum has been used to estimate several interproton distances. The coupling constant and distance data have been used to arrive at sequence-specific sugar geometries and glycosidic torsion angles. The backbone structure has been refined by model building using the FRODO program, employing the sugar geometries and glycosidic torsion angles discussed above. The molecule shows interesting sequence-dependent variations in the structure. The cleavage site of the restriction enzyme XhoI exhibits unique differences in the sugar geometry and backbone torsion angles.

Crystallization and preliminary data of Indian buffalo erythrocyte carbonic anhydrase.

The most abundant anhydrase isoenzyme from the erythrocyte of Indian buffalo has been purified using affinity gel and DEAE-cellulose ion-exchange columns and single crystals suitable for X-ray diffraction studies have been obtained. The unit cell dimensions are a = 46.8 A, b = 104.5 A, c = 60.4 A, beta = 91.2 degrees and the space group is P2(1), with two molecules per asymmetric unit.

Structure, refinement, and function of carbonic anhydrase isozymes: refinement of human carbonic anhydrase I.

The structure of human erythrocyte carbonic anhydrase I has been refined to a final R value of 19% to 2-A resolution by a combination of least squares refinement and model fitting in a three-dimensional graphics display. About 300 solvent atoms have been located bound to the protein molecule. An interesting hydrogen bond network involving Zn2+, the liganded solvent, side chain groups of Thr-199, Glu-106, Thr-7, and His-64 through two solvent molecules have been found that may be important for the catalytic mechanism of the carbonic anhydrase.