Pharmacologic profiling reveals lapatinib as a novel antiviral against SARS-CoV-2 in vitro.

The emergence of SARS-CoV-2 virus has resulted in a worldwide pandemic, but effective antiviral therapies are not widely available. To improve treatment options, we conducted a high-throughput screen to uncover compounds that block SARS-CoV-2 infection. A minimally pathogenic human betacoronavirus (OC43) was used to infect physiologically-relevant human pulmonary fibroblasts (MRC5) to facilitate rapid antiviral discovery in a preclinical model. Comprehensive profiling was conducted on more than 600 compounds, with each compound arrayed across 10 dose points. Our screening revealed several FDA-approved agents that can attenuate both OC43 and SARS-CoV-2 viral replication, including lapatinib, doramapimod, and 17-AAG. Importantly, lapatinib inhibited SARS-CoV-2 RNA replication by over 50,000-fold. Further, both lapatinib and doramapimod could be combined with remdesivir to improve antiviral activity in cells. These findings reveal novel therapeutic avenues that could limit SARS-CoV-2 infection.

Inflammation increases oxidative DNA damage repair and stimulates preneoplastic changes in colons of newborn rats.

Oxidative DNA damage may be a risk factor for development of various pathologies, including malignancy. We studied inflammation triggered modulation of repair activity in the intestines of three weeks old rats injected i.p. with E.coli or S. typhimurium lipopolysaccharides (LPS) at doses of 1, 5 or 10 mg/kg. Subsequent formation in these animals of colonic preneoplastic lesions, aberrant crypt foci (ACF) was also investigated. Five days after LPS administration no differences were observed in repair rate of 1,N(6)-ethenoadenine (εA), 3,N(4)-ethenocytosine (εC) and 8-oxoguanine (8-oxoG) in intestines of these rats, as measured by the nicking assay. However a significant increase in all three repair activities was found within one and two months after S. typhimurium LPS treatment. E. coli LPS significantly increased only the 8-oxoG repair. S. typhimurium LPS stimulated mRNA transcription of pro-inflammatory proteins, lipooxygenase-12 and cyclooxygenase-2, as well as some DNA repair enzymes like AP-endonuclease (Ape1) and εC-glycosylase (Tdg). mRNA level of DNA glycosylases excising εA (MPG) and 8-oxoG (OGG1) was also increased by LPS treatment, but only at the highest dose. Transcription of all enzymes increased for up to 30 days after LPS, and subsequently decreased to the level observed before treatment, with the exception of APE1, which remained elevated even two months after LPS administration. Thus, the repair efficiency of εA, εC and 8-oxoG depends on the availability of APE1, which increases OGG1 and TDG turnover on damaged DNA, and presumably stimulates MPG. One and two months after administration of E. coli or S. typhimurium LPS, the number of aberrant crypt foci in rat colons increased in a dose and time dependent manner. Thus, inflammation stimulates the repair capacity for εA, εC and 8-oxoG, but simultaneously triggers the appearance of preneoplastic changes in the colons. This may be due to increased oxidative stress and imbalance in DNA repair.

A QM-MD simulation approach to the analysis of FRET processes in (bio)molecular systems. A case study: complexes of E. coli purine nucleoside phosphorylase and its mutants with formycin A.

Predicting FRET pathways in proteins using computer simulation techniques is very important for reliable interpretation of experimental data. A novel and relatively simple methodology has been developed and applied to purine nucleoside phosphorylase (PNP) complexed with a fluorescent ligand - formycin A (FA). FRET occurs between an excited Tyr residue (D*) and FA (A). This study aims to interpret experimental data that, among others, suggests the absence of FRET for the PNPF159A mutant in complex with FA, based on novel theoretical methodology. MD simulations for the protein molecule containing D*, and complexed with A, are carried out. Interactions of D* with its molecular environment are accounted by including changes of the ESP charges in S1, compared to S0, and computed at the SCF-CI level. FRET probability W F depends on the inverse six-power of the D*-A distance, R da . The orientational factor 0 < k(2) < 4 between D* and A is computed and included in the analysis. Finally W F is time-averaged over the MD trajectories resulting in its mean value. The red-shift of the tyrosinate anion emission and thus lack of spectral overlap integral and thermal energy dissipation are the reasons for the FRET absence in the studied mutants at pH 7 and above. The presence of the tyrosinate anion results in a competitive energy dissipation channel and red-shifted emission, thus in consequence in the absence of FRET. These studies also indicate an important role of the phenyl ring of Phe159 for FRET in the wild-type PNP, which does not exist in the Ala159 mutant, and for the effective association of PNP with FA. In a more general context, our observations point out very interesting and biologically important properties of the tyrosine residue in its excited state, which may undergo spontaneous deprotonation in the biomolecular systems, resulting further in unexpected physical and/or biological phenomena. Until now, this observation has not been widely discussed in the literature.

Trichinella spiralis infection enhances protein kinase C phosphorylation in guinea pig alveolar macrophages.

To learn more about the signalling pathways involved in superoxide anion production in guinea pig alveolar macrophages, triggered by Trichinella spiralis infection, protein level and phosphorylation of mitogen activated protein (MAP) kinases and protein kinase C (PKC) were investigated. Infection with T. spiralis, the nematode having 'lung phase' during colonization of the host, enhances PKC phosphorylation in guinea pig alveolar macrophages. Isoenzymes beta and delta of PKC have been found significantly phosphorylated, although their location was not changed as a consequence of T. spiralis infection. Neither in macrophages from T. spiralis-infected guinea pig nor in platelet-activating factor (PAF)-stimulated macrophages from uninfected animals, participation of MAP kinases in respiratory burst activation was statistically significant. The parasite antigens seem to act through macrophage PAF receptors, transducing a signal for enhanced NADPH oxidase activity, as stimulating effect of newborn larvae homogenate on respiratory burst was abolished by specific PAF receptor antagonist CV 6209. A suppressive action of T. spiralis larvae on host alveolar macrophage innate immunological response was reflected by diminished protein level of ERK2 kinase and suppressed superoxide anion production, in spite of high level of PKC phosphorylation.

Developmental arrest in Caenorhabditis elegans dauer larvae causes high expression of enzymes involved in thymidylate biosynthesis, similar to that found in Trichinella muscle larvae.

Crude extract specific activities of thymidylate synthase, dUTPase, thymidine kinase and dihydrofolate reductase were high during the development of Caenorhabditis elegans, the dauer larva activities being similar to those previously determined in Trichinella spiralis and T. pseudospiralis muscle larvae (with the exception of thymidine kinase, not detected in Trichinella). High thymidylate synthase expression in developmentally arrested larvae, demonstrated also at the mRNA and protein levels, is in agreement with a global cell cycle arrest of dauer larvae and indicates this unusual cell cycle regulation pattern can be shared by developmentally arrested larvae of C. elegans and the two Trichnella species. Hence, the phenomenon may be characteristic for developmentally arrested larvae of different nematodes, rather than specific for the parasitic Trichinella muscle larvae. Endogenous C. elegans thymidylate synthase was purified and its molecular properties compared with those of the recombinant protein, expression of the latter in E. coli cells confirming the NCBI database sequence identity.

Helicobacter pylori eradication as prevention against chronic peptic ulcer disease in children.

The changes caused by Helicobacter pylori are a slow, progressing inflammatory process developing from several to dozen years. H. pylori infection leads to an inflammatory response in the gastric mucosa with granulocyte infiltrates in an acute form of the inflammation, and lymphocytes, plasmatic, macrophages and eosinophils in a chronic form inducing the development of gastric and duodenal ulcers and gastric cancer in some patients. The frequency and the type of morphological changes in the gastric mucosa were analyzed in children with positive IgG against H. pylori and the incidence of gastric and duodenal ulcers in family members of children examined was evaluated in our study. Gastritis was reported in 68.8% of children with positive IgG against H. pylori. Gastric ulcer was confirmed in 37.1% of families of children included in the study. Duodenal ulcers were found in 22.9% of families. The results obtained, indicate the usefulness of long-term observation and clinical follow-up of children with chronic gastritis of H. pylori ethiology taking into consideration bacterium eradication as prophylaxis of peptic ulceration.

Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression.

The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequenced, allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35,582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Km(app) values desscribing interactions with dUMP (28.8 microM vs. 3.9 microM) and (6RS,alphaS)-N(5,10)-methylenetetrahydrofolate (383 microM vs. 54.7 microM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle.

Peripheral blood lymphocyte population in children infected with Helicobacter pylori.

PURPOSE: Helicobacter pylori infection in children is associated with a chronic inflammatory process of gastric and duodenal mucosa, which may have a various clinical course ranging from asymptomatic and chronic inflammatory condition to gastric ulceration. The immune system may contribute especially to chronic gastric mucosa inflammation. The aim of our study was to assess the levels of peripheral blood T (CD3+, CD4+, CD8+) and B lymphocyte subpopulation (CD19+) in children with Helicobacter pylori infection and to evaluate their relation to degree of antrum mucosa inflammation. MATERIAL AND METHODS: The study was performed in 32 children aged 7-18 years, hospitalized due to dyspeptic symptoms. The endoscopic examination of upper gastrointestinal tract was performed and gastric and duodenal mucosa was estimated in all patients. The endoscopic and histological evaluation of gastric mucosa was performed according to the Sydney System [4]. The urease test (CLO-test-H. pylori) was made to estimate the severity of the infection. RESULTS: Moderate antrum mucosa inflammation was found in 41.2% of the examined. The highest percentage of children (58.8%) presented marked inflammation. No mild inflammation was found in children examined. CONCLUSIONS: No correlation was found between lymphocyte levels and the degree of the inflammatory changes in antrum mucosa. The evaluation of peripheral blood lymphocytes performed in children with Helicobacter pylori infection suggests that T lymphocytes may play a predominant role in this infection.

Interaction of thymidylate synthase with the 5'-thiophosphates, 5'-dithiophosphates, 5'-H-phosphonates and 5'-S-thiosulfates of 2'-deoxyuridine, thymidine and 5-fluoro-2'-deoxyuridine.

New analogs of dUMP, dTMP and 5-fluoro-dUMP, including the corresponding 5'-thiophosphates (dUMPS, dTMPS and FdUMPS), 5'-dithiophosphates (dUMPS2, dTMPS2 and FdUMPS2), 5'-H-phosphonates (dUMP-H, dTMP-H and FdUMP-H) and 5'-S-thiosulfates (dUSSO3, dTSSO3 and FdUSSO3), have been synthesized and their interactions studied with highly purified mammalian thymidylate synthase. dUMPS and dUMPS2 proved to be good substrates, and dTMPS and dTMPS2 classic competitive inhibitors, only slightly weaker than dTMP. Their 5-fluoro congeners behaved as potent, slow-binding inhibitors. By contrast, the corresponding 5'-H-phosphonates and 5'-S-thiosulfates displayed weak activities, only FdUMP-H and FdUSSO3 exhibiting significant interactions with the enzyme, as weak competitive slow-binding inhibitors versus dUMR The pH-dependence of enzyme time-independent inhibition by FdUMP and FdUMPS was found to correlate with the difference in pKa values of the phosphate and thiophosphate groups, the profile of FdUMPS being shifted (approximately 1 pH unit) toward lower pH values, so that binding of dUMP and its analogs is limited by the phosphate secondary hydroxyl ionization. Hence, together with the effects of 5'-H-phosphonate and 5'-S-thiosulfate substituents, the much weaker interactions of the nucleotide analogs (3-5 orders of magnitude lower than for the parent 5'-phosphates) with the enzyme is further evidence that the enzyme's active center prefers the dianionic phosphate group for optimum binding.

Meta-analysis of the relationship between HIV infection and risk for depressive disorders.

OBJECTIVE: Each of 10 published studies investigating the relationship between HIV infection and risk for depressive disorders concluded that HIV-positive individuals are at no greater risk for depression than comparable HIV-negative individuals. This study used meta-analytic techniques to further examine the relationship between depressive disorders and HIV infection. METHOD: Meta-analytic techniques were used to aggregate and reanalyze the data from 10 studies that compared HIV-positive and HIV-negative individuals for rates of major depressive disorder (N=2,596) or dysthymic disorder (N=1,822). RESULTS: The frequency of major depressive disorder was nearly two times higher in HIV-positive subjects than in HIV-negative comparison subjects. On the other hand, findings were inconclusive with regard to dysthymic disorder. Rates of depression do not appear to be related to the sexual orientation or disease stage of infected individuals. CONCLUSIONS: Although the majority of HIV-positive individuals appear to be psychologically resilient, this meta-analysis provides strong evidence that HIV infection is associated with a greater risk for major depressive disorder. Future research should focus on identifying pathways of risk and resilience for depression within this population.

The pyrimidine ring-opened derivative of 1,N6-ethenoadenine is excised from DNA by the Escherichia coli Fpg and Nth proteins.

It was previously shown that 1,N(6)-ethenoadenine (epsilonA) in DNA rearranges into a pyrimidine ring-opened derivative of 20-fold higher mutagenic potency in Escherichia coli (AB1157 lacDeltaU169) than the parental epsilonA (Basu, A. K., Wood, M. L., Niedernhofer, L. J., Ramos, L. A., and Essigmann, J. M. (1993) Biochemistry 32, 12793-12801). We have found that at pH 7.0, the stability of the N-glycosidic bond in epsilondA is 20-fold lower than in dA. In alkaline conditions, but also at neutrality, epsilondA depurinates or converts into products: epsilondA --> B --> C --> D. Compound B is a product of water molecule addition to the C(2)-N(3) bond, which is in equilibrium with a product of N(1)-C(2) bond rupture in epsilondA. Compound C is a deformylated derivative of ring-opened compound B, which further depurinates yielding compound D. Ethenoadenine degradation products are not recognized by human N-alkylpurine-DNA glycosylase, which repairs epsilonA. Product B is excised from oligodeoxynucleotides by E. coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Nth). Repair by the Fpg protein is as efficient as that of 7,8-dihydro-8-oxoguanine when the excised base is paired with dT and dC but is less favorable when paired with dG and dA. Ethenoadenine rearrangement products are formed in oligodeoxynucleotides also at neutral pH at the rate of about 2-3% per week at 37 degrees C, and therefore they may contribute to epsilonA mutations.

[Ormond's fibrosis, bone osteolysis and stomach intramural metastases in the course f low-differentiated prostatic cancer]. / Wlóknienie typu Ormonda, osteoliza kosci oraz sródscienny naciek zoladka w przebiegu niskozróznicowanego raka gruczolu krokowego.

Retroperitoneal fibrosis (Ormond's disease) is rare chronic inflammatory process, that can occur at any age. It is characterised by development of periaortic fibrous mass leading to progressive obstruction of vessels around the abdominal aorta and ureters. In the one third of cases we can find the causes of disease. There are ergotamine abuse, radiation, retroperitoneal surgery or hemorrhage, urine extravasation and response to different cancers. The other cases are idiopathic disease. We report a case of prostate cancer with unique course. The first manifestations of disease were diffuse peritoneal fibrosis and ureteral obstruction leading to bilateral hydronephrosis. Clinical course and histopathology showed idiopathic Ormond's fibrosis. Patient received oral immunosuppressive treatment (prednisolone 1 mg/kg/day + azathioprine 1 mg/kg/day), followed by intravenous methylprednisolone puls (2 g). Treatment also consisted of DJ-stent placement on the left side. On the right side we were unable to overcome the obstruction of ureter. Because of persistent renal failure, thrombocytopenia, DIC and progressive lower back pain we did control MR and CT scan. The CT scans showed multiple osteolytic bone metastases in vertebral column (the sizes of them were between a few millimetres and 1.5 centimetre). Patient died due to renal failure and haemorrhagic diathesis in the course of disseminated cancer of unknown origin. The postmortem examination revealed diffuse peritoneal infiltration surrounding the ureters, intramural ventricular metastases, pulmonary metastases and vertebral metastases. The prostate was only slightly enlarged. Histological and immunohistochemical examinations of prostate showed primary low-differentiated prostate carcinoma (CK/+/, PAP/+/, PSA/+/). Peritoneal, ventricular and bone infiltrations also were metastases from low-differentiated carcinoma of prostate origin (CK/+/, PAP/+/, PSA/-/).

5-Substituted N(4)-hydroxy-2'-deoxycytidines and their 5'-monophosphates: synthesis, conformation, interaction with tumor thymidylate synthase, and in vitro antitumor activity.

Convenient procedures are described for the synthesis of 5-substituted N(4)-hydroxy-2'-deoxycytidines 5a,b,d-h via transformation of the respective 5-substituted 3', 5'-di-O-acetyl-2'-deoxyuridines 1a-c,e-h. These procedures involved site-specific triazolation or N-methylimidazolation at position C(4), followed by hydroxylamination and deblocking with MeOH-NH(3). Nucleosides 5a,b,d-h were selectively converted to the corresponding 5'-monophosphates 6a,b,d-h with the aid of the wheat shoot phosphotransferase system. Conformation of each nucleoside in D(2)O solution, deduced from (1)H NMR spectra and confirmed by molecular mechanics calculations, showed the pentose ring to exist predominantly in the conformation S (C-2'-endo) and the N(4)-OH group as the cis rotamer. Cell growth inhibition was studied with two L5178Y murine leukemia cell lines, parental and 5-fluoro-2'-deoxyuridine (FdUrd)-resistant, the latter 70-fold less sensitive toward FdUrd than the former. With FdUrd-resistant L5178Y cells, 5-fluoro-N(4)-hydroxy-2'-deoxycytidine (5e) caused almost 3-fold stronger growth inhibition than FdUrd; 5e was only some 3-fold weaker growth inhibitor of the resistant cells than of the parental cells. Thymidylate synthase inhibition was studied with two forms of the enzyme differing in sensitivities toward 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), isolated from parental and FdUrd-resistant L1210 cell lines. All N(4)-hydroxy-dCMP (6a,b,d-h) and dUMP analogues studied were competitive vs dUMP inhibitors of the enzyme. Analogues 6b,d-h and 5-hydroxymethyl-dUMP, similar to N(4)-hydroxy-dCMP (6a) and FdUMP, were also N(5), N(10)-methylenetetrahydrofolate-dependent, hence mechanism-based, slow-binding inhibitors. 5-Chloro-dUMP, 5-bromo-dUMP, and 5-iodo-dUMP, similar to dTMP, did not cause a time-dependent inactivation of the enzyme. Instead, they behaved as classic inhibitors of tritium release from [5-(3)H]dUMP. 5-Bromo-dUMP and 5-iodo-dUMP showed substrate activity independent of N(5), N(10)-methylenetetrahydrofolate in the thymidylate synthase-catalyzed dehalogenation reaction. The =N-OH substituent of the pyrimidine C(4) prevented the enzyme-catalyzed release from the C(5) of Br(-) and I(-) (the same shown previously for H(+)). While FdUMP and 6a showed a higher affinity and greater inactivation power with the parental cell than FdUrd-resistant cell enzyme, an opposite relationship could be seen with 5-hydroxymethyl-dUMP.

Relapse patterns among adolescents treated for chemical dependency.

This study examined levels, timing, and patterns of posttreatment alcohol and drug use in adolescents (n = 113) during the 12 months following completion of primary treatment. Data were collected from clinical records, random urine screens, and interviews with the adolescents and their parents. In all, 61.1% relapsed to pretreatment levels of use during the 12 months after treatment, 79.6% of the males and 59.3% of the females. Of those with alcohol as the drug of dependence, 45.9% relapsed to pretreatment levels of use in 12 months. Likewise, 75.0% marijuana users, 70.6% combined alcohol and marijuana users, and 50.2% other drug users relapsed to pretreatment levels of use in the 12 months. Relapse curves are presented to demonstrate the specific timing and patterns of relapse. Implications for primary treatment and aftercare are discussed.

Crystal structures of rat thymidylate synthase inhibited by Tomudex, a potent anticancer drug.

Two crystal structures of rat thymidylate synthase (TS) complexed with dUMP and the anticancer drug Tomudex (ZD1694) have been determined to resolutions of 3.3 and 2.6 A. Tomudex is one of several new antifolates targeted to TS and the first to be approved for clinical use. The structures represent the first views of any mammalian TS bound to ligands and suggest that the rat protein undergoes a ligand-induced conformational change similar to that of the Escherichia coli protein. Surprisingly, Tomudex does not induce the "closed" conformation in rat TS that is seen on binding to E. coli TS, resulting in inhibitor atoms that differ in position by more than 1.5 A. Several species-specific differences in sequence may be the reason for this. Phe 74 shifts to a new position in the rat complex and is in van der Waals contact with the inhibitor, while in the E. coli protein the equivalent amino acid (His 51) hydrogen bonds to the glutamate portion of the inhibitor. Amino acids Arg 101, Asn 106, and Met 305 make no contacts with the inhibitor in the open conformation, unlike the equivalent residues in the E. coli protein (Thr 78, Trp 83, and Val 262). dUMP binding is similar in both proteins, except that there is no covalent adduct to the active site cysteine (Cys 189) in the rat structures. Two insertions in the rat protein are clearly seen, but the N-termini (residues 1-20) and C-termini (residues 301-307) are disordered in both crystal forms.

Military preventive medicine support: the Balkan experience.

Preventive medicine (PVNTMED) support to deployed forces is as varied as the circumstances for force deployment. The Bosnia-Herzegovina deployment of the 1st Armored Division as part of the Dayton Peace Accords Implementation Forces proved to be no exception to this premise. PVNTMED units, both in the field and at the U.S. Army-Europe support base, were challenged to provide mission support under significant mobility restrictions and in arenas of public health practice not previously thought to be of tactical significance, specifically environmental pollution. New to this operation was the deployment of a Theater Army Medical Laboratory with a mission to assist deployed PVNTMED units with the capability to rapidly diagnose infectious disease agents and provide an expanded array of environmental monitoring support. Vector-borne diseases were also a threat to health, and an innovative base camp sanitation assessment and reporting system was created to alert leaders to the risk of disease transmission to soldiers.