Multidrug-Resistant Tuberculosis-Diagnostic Procedures and Treatment of Two Beijing-like TB Cases.

The Beijing/W genotype is one of the major molecular families of Mycobacterium tuberculosis complex (MTBC), responsible for approximately 50% of tuberculosis (TB) cases in Far East Asia and at least 25% of TB cases globally. Studies have revealed that the Beijing genotype family is associated with a more severe clinical course of TB, increased ability to spread compared to other genotypes, and an unpredictable response to treatment. Based on the profile of spacers 35-43 in the Direct Repeat (DR) locus of the MTBC genome determined by spoligotyping, classical (typical) and modern (Beijing-like) clones can be identified within the Beijing family. While the modern and ancient Beijing strains appear to be closely related at the genetic level, there are marked differences in their drug resistance, as well as their ability to spread and cause disease. This paper presents two cases of drug-resistant tuberculosis caused by rare mycobacteria from the Beijing family: the Beijing 265 and Beijing 541 subtypes. The genotypes of isolated strains were linked with the clinical course of TB, and an attempt was made to initially assess whether the Beijing subtype can determine treatment outcomes in patients.

Nontuberculous mycobacteria strains isolated from patients between 2013 and 2017 in Poland. Our data with respect to the global trends.

INTRODUCTION: During the last decades the prevalence of NTM infections has increased, especially in developed countries. The aim of the study was to provide an overview on all NTM isolated from clinical samples in Poland between 2013 and 2017. MATERIAL AND METHODS: The study comprised 2799 clinical specimens, mostly respiratory accessed in the reference laboratory of National Tuberculosis and Lung Diseases Research Institute in Warsaw and in the Wielkopolska Center of Pulmonology and Thoracic Surgery, Poland, 2013-2017. RESULTS: During the study period 35 species of NTM were isolated . The number of isolates increased almost 1.6-fold: from 420 in 2013 to 674 in 2017. M. kansasii, M. avium, M. xenopi, M. gordonae and M. intracellulare were the most common species. This NTM pattern was rather stable over the time. If the aggregated amount of all MAC species was taken into account they dominated over M. kansasii from 2015. M. avium and M. intracellulare were more often isolated from women, while M. kansasii, M. gordonae and M. xenopi predominated in men. Men and women were infected almost with the same frequency. In older patients 65+ women were in majority, quite opposite to those aged 25 to 64 years. CONCLUSION: In Poland, like in other countries increased the frequency of isolated NTM. M. kansasii and M. avium were the most frequently identified species from clinical samples. Men and women were infected with NTM with the same frequency.

Recommendations for the management of tuberculosis in children - KOMPASS TB. Part 1: Tuberculosis prevention.

Since the second half of the 20th century the incidence of tuberculosis has been declining in Poland. Despite this, current epidemiological data still support the need for the continued mass BCG vaccination in Poland in the near future. Apart from the protection against severe hematogenous forms of tuberculosis, vaccination lowers the risk of infection with Mycobacterium tuberculosis. Primary and acquired immunodeficiency, including immunity disorders associated with an ongoing treatment, are contraindications to BCG vaccination. The most common adverse effects following BCG vaccination are reactions at the site of injection and in regional lymph nodes, which usually does not require treatment. Methods of tuberculosis prevention, particularly recommended in low-incidence countries, include: diagnostic investigations of patients who had contacts with pulmonary tuberculosis as well as an active detection and treatment of latent Mycobacterium tuberculosis infection. Latent tuberculosis infection can be identified on the basis of positive results of the tuberculin skin test or interferon-gamma release assays after the active disease has been ruled out. This condition does require prophylactic treatment.

From molecular phylogeny towards differentiating pharmacology for NMDA receptor subtypes.

In order to decode the roles that N-methyl-D-aspartate (NMDA) receptors play in excitatory neurotransmission, synaptic plasticity, and neuropathologies, there is need for ligands that differ in their subtype selectivity. The conantokin family of Conus peptides is the only group of peptidic natural products known to target NMDA receptors. Using a search that was guided by phylogeny, we identified new conantokins from the marine snail Conus bocki that complement the current repertoire of NMDA receptor pharmacology. Channel currents measured in Xenopus oocytes demonstrate conantokins conBk-A, conBk-B, and conBk-C have highest potencies for NR2D containing receptors, in contrast to previously characterized conantokins that preferentially block NR2B containing NMDA receptors. Conantokins are rich in γ-carboxyglutamate, typically 17-34 residues, and adopt helical structure in a calcium-dependent manner. As judged by CD spectroscopy, conBk-C adopts significant helical structure in a calcium ion-dependent manner, while calcium, on its own, appears insufficient to stabilize helical conformations of conBk-A or conBk-B. Molecular dynamics simulations help explain the differences in calcium-stabilized structures. Two-dimensional NMR spectroscopy shows that the 9-residue conBk-B is relatively unstructured but forms a helix in the presence of TFE and calcium ions that is similar to other conantokin structures. These newly discovered conantokins hold promise that further exploration of small peptidic antagonists will lead to a set of pharmacological tools that can be used to characterize the role of NMDA receptors in nervous system function and disease.

Stapling mimics noncovalent interactions of γ-carboxyglutamates in conantokins, peptidic antagonists of N-methyl-D-aspartic acid receptors.

Conantokins are short peptides derived from the venoms of marine cone snails that act as antagonists of the N-methyl-D-aspartate (NMDA) receptor family of excitatory glutamate receptors. These peptides contain γ-carboxyglutamic acid residues typically spaced at i,i+4 and/or i,i+7 intervals, which by chelating divalent cations induce and stabilize helical conformation of the peptide. Introduction of a dicarba bridge (or a staple) can covalently stabilize peptide helicity and improve its pharmacological properties. To test the hypothesis that stapling can effectively replace γ-carboxyglutamic acid residues in stabilizing the helical conformation of conantokins, we designed, synthesized, and characterized several stapled analogs of conantokin G (conG), with varying connectivities in terms of staple length and location along the face of the α-helix. NMR studies confirmed that the ring-closing metathesis reaction yielded a single product with the Z configuration of the olefinic bond. Based on circular dichroism and molecular modeling, the stapled analogs exhibited significantly enhanced helicity compared with the native peptide in a metal-free environment. Stapling i,i+4 was benign with respect to effects on in vitro and in vivo pharmacological properties. One analog, namely conG[11-15,S(i,i+4)S(8)], blocked NR2B-containing NMDA receptors with IC(50) = 0.7 µm and provided significant protection in the 6-Hz psychomotor model of pharmacoresistant epilepsy in mice. Remarkably, unlike native conG, conG[11-15,S(i,i+4)S(8)] produced no behavioral motor toxicity. Our results extend the applications of peptide stapling to helical peptides with extracellular targets and provide a means for engineering conantokins with improved pharmacological properties.

ATP and its N6-substituted analogues: parameterization, molecular dynamics simulation and conformational analysis.

In this work we used a combination of classical molecular dynamics and simulated annealing techniques to shed more light on the conformational flexibility of 12 adenosine triphosphate (ATP) analogues in a water environment. We present simulations in AMBER force field for ATP and 12 published analogues [Shah et al. (1997) Proc Natl Acad Sci USA 94: 3565-3570]. The calculations were carried out using the generalized Born (GB) solvation model in the presence of the cation Mg(2+). The ion was placed at a close distance (2 Å) from the charged oxygen atoms of the beta and gamma phosphate groups of the -3 negatively charged ATP analogue molecules. Analysis of the results revealed the distribution of inter-proton distances H8-H1' and H8-H2' versus the torsion angle ψ (C4-N9-C1'-O4') for all conformations of ATP analogues. There are two gaps in the distribution of torsion angle ψ values: the first is between -30 and 30 degrees and is described by cis-conformation; and the second is between 90 and 175 degrees, which mostly covers a region of anti conformation. Our results compare favorably with results obtained in experimental assays [Jiang and Mao (2002) Polyhedron 21:435-438].

[Diagnostic significance of tuberculin skin test in BCG vaccinated population]. / Znaczenie diagnostyczne próby tuberkulinowej w populacji szczepionej BCG.

UNLABELLED: There is some data on the influence of the BCG vaccination on the TST (tuberculin skin test) but most of them are focused on the population vaccinated only one time. The aim of the study was to describe the relation between vaccination scheme and TST in polish population. MATERIAL AND METHODS: TST was performed in 114 patient with active, culture confirmed tuberculosis and 100 medical student at the beginning of the study as a control group. RESULTS: In our investigation there was no statistically important differences of the TST size between Tb patients and control group. CONCLUSION: In polish population it is not possible to estimate the cutoff value of TST size that could be used to differentiate between TB infection and reaction to vaccination.

Phosphorylation and ATP-binding induced conformational changes in the PrkC, Ser/Thr kinase from B. subtilis.

Recent studies on the PrkC, serine-threonine kinase show that that the enzyme is located at the inner membrane of endospores and is responsible for triggering spore germination. The activity of the protein increases considerably after phosphorylation of four threonine residues placed on the activation loop and one serine placed in the C-terminal lobe of the PrkC. The molecular relationship between phosphorylation of these residues and enzyme activity is not known. In this work molecular dynamics simulation is performed on four forms of the protein kinase PrkC from B. subtilis-phosphorylated or unphosphorylated; with or without ATP bound-in order to gain insight into phosphorylation and ATP binding on the conformational changes and functions of the protein kinase. Our results show how phosphorylation, as well as the presence of ATP, is important for the activity of the enzyme through its molecular interaction with the catalytic core residues. Three of four threonine residues were found to be involved in the interactions with conservative motifs important for the enzyme activity. Two of the threonine residues (T167 and T165) are involved in ionic interactions with an arginine cluster from alphaC-helix. The third residue (T163) plays a crucial role, interacting with His-Arg-Asp triad (HRD). Last of the threonine residues (T162), as well as the serine (S214), were indicated to play a role in the substrate recognition or dimerization of the enzyme. The presence of ATP in the unphosphorylated model induced conformational instability of the activation loop and Asp-Phe-Gly motif (DFG). Based on our calculations we put forward a hypothesis suggesting that the ATP binds after phosphorylation of the activation loop to create a fully active conformation in the closed position.

Cooccupancy of the outer vestibule of voltage-gated sodium channels by micro-conotoxin KIIIA and saxitoxin or tetrodotoxin.

The guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX) are classic ligands of voltage-gated sodium channels (VGSCs). Like TTX and STX, micro-conotoxin peptides are pore blockers but with greater VGSC subtype selectivity. micro-Conotoxin KIIIA blocks the neuronal subtype Na(V)1.2 with nanomolar affinity and we recently discovered that KIIIA and its mutant with one fewer positive charge, KIIIA[K7A], could act synergistically with TTX in a ternary peptide x TTX x Na(V) complex. In the complex, the peptide appeared to trap TTX in its normal binding site such that TTX could not readily dissociate from the channel until the peptide had done so; in turn, the presence of TTX accelerated the rate at which peptide dissociated from the channel. In the present study we examined the inhibition of Na(V)1.2, exogenously expressed in Xenopus oocytes, by STX (a divalent cation) and its sulfated congener GTX2/3 (with a net +1 charge). Each could form a ternary complex with KIIIA and Na(V)1.2, as previously found with TTX (a monovalent cation), but only when STX or GTX2/3 was added before KIIIA. The KIIIA x alkaloid x Na(V) complex was considerably less stable with STX than with either GTX2/3 or TTX. In contrast, ternary KIIIA[K7A] x alkaloid x Na(V) complexes could be formed with either order of ligand addition and were about equally stable with STX, GTX2/3, or TTX. The most parsimonious interpretation of the overall results is that the alkaloid and peptide are closely apposed in the ternary complex. The demonstration that two interacting ligands ("syntoxins") occupy adjacent sites raises the possibility of evolving a much more sophisticated neuropharmacology of VGSCs.

Conantokin-Br from Conus brettinghami and selectivity determinants for the NR2D subunit of the NMDA receptor.

Conantokins are venom peptides from marine cone snails that are NMDA receptor antagonists. Here, we report the characterization of a 24 AA conantokin from Conus brettinghami Coomans , H. E. , Moolenbeek , R. G. and Wils , E. ( 1982 ) Basteria 46 ( 1/4 ), 3 - 67 , conantokin-Br (con-Br), the first conantokin that does not have the conserved glutamate residue at position 2. Molecular modeling studies suggest that con-Br has a helical structure between residues 2-13. In contrast to other characterized conantokins, con-Br has a high potency for NMDA receptors with NR2D subunits. To identify determinants for NR2D potency, we synthesized chimeras of con-Br and conantokin-R (con-R); the latter has a approximately 30-fold lower potency for the NR2D subtype. The characterization of two reciprocal chimeras (con-Br/R and con-R/Br), comprising the first 9-10 N-terminal AAs of each conantokin followed by the corresponding C-terminal AAs of the other conantokin demonstrates that determinants for NR2D selectivity are at the N-terminal region. Additional analogues comprising 1-3 amino acid substitutions from each peptide into the homologous region of the other led to the identification of a key determinant; a Tyr residue in position 5 increases potency for NR2D, while Val at this locus causes a decrease. The systematic definition of key determinants in the conantokin peptides for NMDA receptor subtype selectivity is an essential component in the development of conantokin peptides that are highly selective for each specific NMDA receptor subtype.

Structurally minimized mu-conotoxin analogues as sodium channel blockers: implications for designing conopeptide-based therapeutics.

Disulfide bridges that stabilize the native conformation of conotoxins pose a challenge in the synthesis of smaller conotoxin analogues. Herein we describe the synthesis of a minimized analogue of the analgesic mu-conotoxin KIIIA that blocks two sodium channel subtypes, the neuronal Na(V)1.2 and skeletal muscle Na(V)1.4. Three disulfide-deficient analogues of KIIIA were initially synthesized in which the native disulfide bridge formed between either C1-C9, C2-C15, or C4-C16 was removed. Deletion of the first bridge only slightly affected the peptide's bioactivity. To further minimize this analogue, the N-terminal residue was removed and two nonessential serine residues were replaced by a single 5-amino-3-oxapentanoic acid residue. The resulting "polytide" analogue retained the ability to block sodium channels and to produce analgesia. Until now, the peptidomimetic approach applied to conotoxins has progressed only modestly at best; thus, the disulfide-deficient analogues containing backbone spacers provide an alternative advance toward the development of conopeptide-based therapeutics.