Dry eye among medical students before and during COVID-19.

Purpose: Dry eye is a multifactorial disease of the ocular surface. It showed an increased incidence during the pandemic situation, which may be due to long hours of exposure to electronic gadgets. We aimed to find the prevalence of dry eye disease among medical students during the coronavirus disease 2019 (COVID-19) pandemic and pre-pandemic periods. Methods: This was a cross-sectional study conducted in a tertiary care teaching institute. This was an institution-based, cross-sectional study conducted among medical students. A modified Ocular Surface Disease Index (OSDI) questionnaire was used to find the severity and prevalence of dry eye disease. Considering 95% confidence interval (CI) and prevalence as 50%, the calculated sample size was 271. Online responses were collected and entered in an Excel sheet. The Chi-square test, univariate and multivariate logistic regression were used for statistical analysis. Results: Data were collected from 271 medical students; the prevalence of dry eye disease was 41.5 and 55.19 during the pre-pandemic and pandemic periods, respectively. There was a significant rise in dry eye disease cases during the pandemic when compared to pre-pandemic period (P < 0.05). The odds of getting dry eye disease were 1.7 times more during the pandemic than pre-pandemic. Conclusion: The lockdown situation during the pandemic forced people to use electronic gadgets for work, recreation, and academics. Prolonged screen time predisposes to the development of dry eye disease.

COMPLEX ANALYSIS OF THE ROLE OF CYTOKINE GENE POLYMORPHISMS AS PROGNOSTIC FACTOR OF THE RISK OF PLASMA CELL MYELOMA IN PERSONS SUFFERED AFTER THE CHORNOBYL NPP ACCIDENT. / KOMPLEKSNYI ANALIZ ROLI POLIMORFIZMU GENIV TsYTOKINIV IaK PROGNOSTYChNOGO ChYNNYKA RYZYKU PLAZMOKLITYNNOÏ MIIeLOMY U OSIB, POSTRAZhDALYKh VNASLIDOK AVARIÏ NA ChORNOBYL'S'KII AES.

OBJECTIVE: to provide a comparative characterization of the prevalence of polymorphic variants of cytokine genes in plasma cell myeloma (PCM) patients suffered after the Chornobyl disaster and patients who were in contact with ionizing radiation within the natural radiation background, based on comparison with population controls to determine their contribution as genetic markers of disease risk. MATERIALS AND METHODS: Molecular genetic studies of polymorphism of cytokine genes (TNF-α, TGF-ß1, IL-6, IL-10, IFN-γ) and complex frequency analysis of occurrence in three-, four-, and five-locus combinations of their allelic variants as prognostic markers of the risks of plasma cell myeloma was carried out in 102 patients - 56 victims of the Chornobyl nuclear power plant accident and 46 patients irradiated within the limits of the natural radiation background, in comparison with the control group (364 practically healthy people, residents of the Central geno-geographical region of Ukraine). RESULTS: The same probable increase in the prevalence of the TGF-ß genotype codon10 T/T of the TGF-ß1 gene was established in the groups of patients irradiated after the Chornobyl NPP accident and non-irradiated patients. In patients with plasma cell myeloma a protective effect for IL-10 -1082 A/G and an association with the risk of disease occurrence for IL-10 -1082 G/G were determined. CONCLUSION: Probable difference in the frequency of the TGF-ß1 genotype codon10 T/T of the TGF-ß1 gene in the observed groups relative to the control group provides grounds for considering this single-nucleotide polymorphism of the TGF-ß1 gene as an immunogenetic factor of predisposition to the development of PCM independent of exogenous factors. The study of the contribution of multigene combinations of «gene-gene¼ interaction indicates their role in the mechanisms of plasma cell myeloma occurrence and confirms the presence of an additive interaction.

Biotinylated chitosan macromolecule based nanosystems: A review from chemical design to biological targets.

World Health Organization estimates that 30-50% of cancers are preventable by healthy lifestyle choices, early detection and adequate therapy. When the conventional therapeutic strategies are still regulated by the lack of selectivity, multidrug resistance and severe toxic side effects, nanotechnology grants a new frontier for cancer management since it targets cancer cells and spares healthy tissues. This review highlights recent studies using biotin molecule combined with functional nanomaterials used in biomedical applications, with a particular attention on biotinylated chitosan-based nanosystems. Succinctly, this review focuses on five areas of recent advances in biotin engineering: (a) biotin features, (b) biotinylation approaches, (c) biotin functionalized chitosan based nanosystems for drug and gene delivery functions, (d) diagnostic and theranostic perspectives, and (e) author's inputs to the biotin-chitosan based tumour-targeting drug delivery structures. Precisely engineered biotinylated-chitosan macromolecules shaped into nanosystems are anticipated to emerge as next-generation platforms for treatment and molecular imaging modalities applications.

EFFICIENCY OF BONE MARROW PRECURSOR CELL COLONY-FORMING AS A PREDICTOR OF DISEASE COURSE IN PLASMA CELL MYELOMA PATIENTS WITH A HISTORY OF RADIATION EXPOSURE. / EFEKTYVNIST' KOLONIIeUTVORENNIa KLITYN-POPEREDNYKIV KISTKOVOGO MOZKU, IaK PROGNOSTYChNYI FAKTOR PEREBIGU PLAZMOKLITYNNOÏ MIIeLOMY U OSIB Z RADIATsIINYM ANAMNEZOM.

OBJECTIVE: Assessment of role of the bone marrow colony-forming efficiency in plasma cell myeloma patients at different stages of treatment as a prognostic criterion for the disease course. MATERIALS AND METHODS: The colony forming efficiency (CFE) was assayed in stage I-II plasma cell myeloma (PCM)patients (n = 37) aged 42-73, namely in patients survived after the Chornobyl NPP accident (n = 21) and persons notexposed to ionizing radiation (n = 16). There were 11 males exposed to ionizing radiation and having got stage I PCM,9 males and 3 females exposed and having got stage II PCM, 3 males and 3 females not exposed and having got stageI PCM, 6 males and 2 females not exposed and having got stage II PCM. Healthy persons (n = 20) were included in thecontrol group. RESULTS: Number of the bone marrow (BM) granulocyte-macrophage colony-forming units (CFU-GM) in both exposedand not exposed PCM patients depended on a disease stage. CFU-GM was (16.7 ± 1.2) in the stage I PCM patients vs.(11.1 ± 1.1) in the stage II PCM ones both being lower (p < 0.05) compared to control (64.5 ± 2.2). Changes in cluster formation were similar, i.e. (37.7 ± 1.6) and (19.4 ± 1.3) correspondingly in the stage I and stage II PCM patients.Respective values in control were (89.8 ± 3.6). The CFE in stage I and stage II PCM patients at the time of diagnosiswas lower (5.7 ± 1.5 and 2.4 ± 1.1 respectively) vs. control (39.5 ± 1.51, p < 0.05), but has increased in remission upto (29. 6 ± 1.8) and (13.8 ± 1.2) respectively. There was no difference at that between the irradiated and non-irradiated patients. Number of the fibroblast colony-forming units (CFU-F) in the stage I and stage II PCM patients duringdiagnosis, namely (43.9 ± 5.4) and (22.5 ± 3.7), was lower (p < 0.05) vs. control (110.5 ± 4.9). Upon reaching remission the CFU-F value increased significantly (p < 0.05), reaching (87.4 ± 4.2) and (55.6 ± 2.7) correspondingly in thestage I and stage II PCM patients. CONCLUSION: Dependence of the BM cell CFE on the stage of PCM and presence or absence of remission was established. Prognostic value of the CFE of BM CFU-GM in terms of life span of patients was shown (Ro Spearm = 0.39,p < 0.02), namely in case of CFE > 20 before the polychemotherapy administration the life span of PCM patients wassignificantly longer vs. cases of CFE < 20.

[Current aspects of the treatment of urogenital atrophy (based on the consensus of britain society of menopause)].

The symptoms of genitourinary syndrome of menopause are considered as typical for late menopausal period. However, these symptoms are increasingly diagnosed in perimenopausal and early menopausal period. Women seldom seek medical care, since autonomic menopausal symptoms are usually more bothersome. In many cases, doctors are not sure in necessity of any hormonal replacement. Moreover, a confusion still exists between systemic hormone replacement therapy (HRT) and local estrogen preparations. Besides moisturizers and local intravaginal estrogens, novel treatment modalities have emerged that extend therapeutic armamentarium.

IMMUNOGENETIC AND PHARMACOCHEMICAL CHARACTERIZATION OF THE ABO SYSTEM GLYCOPROTEIN PROPERTIES AS CRITERIA OF INDIVIDUAL SENSITIVITY TO ANTITUMOR AGENT BORTEZOMIB IN THE PLASMA CELL MYELOMA PATIENTS. / IMUNOGENETYChNYY̆ TA FARMAKOKhIMIChNYY̆ ANALIZ VLASTYVOSTEY̆ GLIKOPROTEÏNIV SYSTEMY AVO IaK KRYTERIÏV INDYVIDUAL'NOÏ ChUTLYVOSTI ShchODO PROTYPUKhLYNNOGO PREPARATU BORTEZOMIBU U KhVORYKh NA PLAZMOKLITYNNU MIIeLOMU.

OBJECTIVE: Experimental study of the effect profile of bortezomib in the plasma cell myeloma (PCM) patients depend- ing on a specific phenotype carrier state and a pharmacochemical characteristics of ABO system glycoproteins. MATERIALS AND METHODS: The research was conducted on the 104 PCM patients, including the Chornobyl NPP acci- dent survivors (n = 49) and 65 study subjects in the comparison group. Immunogenetic criteria for positive response to the applied treatment protocols were issued according to the duration of remission, absence of infectious com- plications, and evidence of chronic renal failure as a disease complication. RESULTS: Possibility of glycoproteins A and B participation in the formation of human biological individuality at a level of protein-protein interaction with antineoplastic drug bortezomib, which is widely used in cancer management prac- tice, in particular in the PCM treatment is considered. The glycoprotein B was shown being a selective target for borte- zomib, slowing down the recognition and interaction of antigen B with monoclonal anti-B antibody, while the agglu- tination period lengthens at that by 66 %. Assumption that the formation of bortezomib complex with glycoprotein B provides a background for interaction with the key reaction of proteasome 26S inhibition, which to some extent con- tributes to the drug effect retardation was confirmed through the quantum-chemical calculations. Equilibrium is shift- ed toward the main reaction leading to a higher drug efficacy in patients with blood groups O (I) and A (II). CONCLUSIONS: Since the complexation occurs predominantly in alkaline medium the administration of drugs with alkaline reaction should be restricted for at least round the clock after administration of bortezomib according to its half-life in plasma in patients with B (III) blood group and chronic renal failure.

Assessment of response to imatinib therapy in patients with chronic myeloid leukemia with e13a2 and e14a2 transcripts of BCR/ABL1 gene. / OTsINKA VIDPOVIDI NA TERAPIIu IMATYNIBOM U PATsIIeNTIV Z KhRONIChNOIu MIIeLOIDNOIu LEYKEMIIeIu Z E13A2 TA E14A2 TRANSKRYPTAMY GENA BCR/ABL1.

OBJECTIVE: Assess the influence of e13a2 and e14a2 transcripts of BCR/ABL1 gene on the efficiency of imatinib ther apy in patients with chronic myeloid leukemia. MATERIALS AND METHODS: We examined 508 patients with the chronic phase of chronic myeloid leukemia without radi ation in anamnesis as well as 13 patients with the similar diagnosis and with confirmed presence of radiation expo sure due to the Chornobyl Nuclear Power Plant accident. RESULTS: No significant differences in hematologic parameters, rate of additional chromosomal aberrations and f vari ant translocations were observed between patients with е13а2 and е14а2 transcript. Cumulative probability of com plete cytogenetic response did not differ in patients with е13а2 and е14а2 transcript and was 76 and 80 % respec tively (р = 0,981). Median of achieving a complete cytogenetic response was 20 months in both patient groups. Significantly more patients with e14a2 transcript compared to patients with e13a2 achieved major molecular response by 12 month of therapy (61.5 % versus 23.0 %, p = 0.016). The higher incidence of deep molecular response by 24 month of therapy was revealed in this group (38.7 % versus 6.25 %, p = 0.018). The overall survival and pro gression free survival rates were not statistically different between two groups with different transcripts. However, the rate of event free survival was statistically lower for the patients with e13a2 transcript compared to the ones with e14a2 transcript (51 % versus 62.0 %, p = 0.039). The number of primary resistant patients was 40 % regardless of the transcript expressed. A significant prevalence in incidence either of lost complete cytogenetic response or fail ure of the major molecular response was shown in patients with e13a2 transcript compared to patients with e14a2 transcripts (43.5 % versus 24.8 %, p = 0.015). CONCLUSION: Imatinib therapy is more effective for CML patients with e14a2 transcript compared to patients with e13a2 transcript expression. The transcript e13a2can be viewed as a adverse prognostic factor for imatinib therapy of chronic myeloid leukemia.

The efficiency of tyrosine kinase inhibitor therapy in patients with chronic myeloid leukemia exposed to ionizing radiation due to the Chornobyl nuclear power plant accident. / Efektyvnist'' terapii' ingibitoramy tyrozynkinaz pacijentiv z hronichnoju mijeloi'dnoju lejkemijeju, jaki zaznaly vplyvu ionizujuchogo vyprominjuvannja vnaslidok avarii' na Chornobyl''s''kij AES.

Objective. To study the efficiency of tyrosine kinase inhibitors (TKI) therapy in patients with chronic myeloid leukemia (CML) exposed to ionizing radiation due to the Chornobyl NPP accident, based on the data of cytogenetic and molecular monitoring. Material and methods. 29 CML patients with confirmed radiation exposure due to Chornobyl NPP accident were examined. Of these, 20 patients were treated with imatinib; 103 patients with CML without radiation history treated with TKI were a comparison group. Cytogenetic and molecular genetic disturbances before and on the different stage of TKI therapy were analysed. Results. Additional chromosomal abnormalities as well as special pattern of BCR/ABL transcripts were not revealed in CML patients exposed to ionizing radiation. Complete cytogenetic response (CCR) was shown in 50 and 48.5 % of patients from study and comparison group, respectively. Major molecular response (MMR) was achieved in 20 % of patients with radiation exposure in anamnesis and in 27.6 % of patients from comparison group. The vast majority of CCR and MMR was reached in patients with the pretreatment term up to 6 months, when imatinib was used as a first line therapy. There were less cases of primary imatinib resistance in the same group of patients. In CML patients who had a history of radiation exposure, secondary resistance developed more frequently than in the comparison group and was 25 %. Conclusion. Laboratory monitoring based on the registration of CCR and MMR demonstrated high efficiency of TKI in the CML treatment of patients, exposed due to Chornobyl accident. Extension of pretreatment term leads to the loss of TKI therapy efficiency and increases the likelihood of primary resistance. CML patients exposed to ionizing radiation develop secondary resistence more often than CML patients without radiation exposure in anamnesis.

Current concepts on cardiovascular stent devices.

Coronary artery stents used for the treatment of patients with coronary artery disease develop the practice of interventional cardiology after they were first introduced in the mid-1980s. Since then, with dozens of companies involved in the development of new and innovative anti-restenotic drugs, polymeric coatings and stent platforms has made significant progress in this area. Today, the challenge is the conception of the "ideal" coronary stent designed to respond to the patient health difficulty. In this context, the literature in the field is quite dynamic and successful. The aim of this article is to provide a systematic review on the interdisciplinary field literature of the evolution of these medical devices by describing the current status, importance and different types of stents used in clinical practice. After the presentation of cardiovascular problems associated to stenting therapy, the authors describe the bare metal stents, the generations of drug eluting stents and the future in progress directions regarding: the stents based on biodegradable/bioresorbable polymers, polymer-free metal platforms, fully biodegradable scaffolds, as well as drug delivery mediated by stent-targeted magnetic nanoparticles.

A Salmonella Enteritidis hilAssrAfliG deletion mutant is a safe live vaccine strain that confers protection against colonization by Salmonella Enteritidis in broilers.

Consumption of contaminated poultry meat is an important cause of Salmonella infections in humans. Therefore, there is a need for control methods that protect broilers from day-of-hatch until slaughter age against infection with Salmonella. Colonization-inhibition, a concept in which a live Salmonella strain is orally administered to day-old chickens and protects against subsequent challenge, can potentially be used as control method. In this study, the safety and efficacy of a Salmonella Enteritidis ΔhilAssrAfliG strain as a colonization-inhibition strain for protection of broilers against Salmonella Enteritidis was evaluated. After administration of the Salmonella Enteritidis ΔhilAssrAfliG strain to day-old chickens, this strain could not be isolated from the gut, internal organs or faeces after 21 days of age. In addition, administration of this strain to one-day-old broiler chickens decreased faecal shedding and caecal and internal organ colonization of a Salmonella Enteritidis challenge strain administered one day later using a seeder bird model. To our knowledge, this is the first report of an attenuated Salmonella strain for which both the safety and efficacy has been shown in long-term experiments (until slaughter age) in broiler strain can potentially be used as a live colonization-inhibition strain for controlling Salmonella Enteritidis infections in broilers.

The significance of galectin-3 as a new basal cell marker in prostate cancer.

Prostate cancer may originate from distinct cell types, resulting in the heterogeneity of this disease. Galectin-3 (Gal-3) and androgen receptor (AR) have been reported to play important roles in the progression of prostate cancer, and their heterogeneous expressions might be associated with different cancer subtypes. Our study found that in various prostate cancer cell lines Gal-3 expression was always opposite to AR expression and other luminal cell markers but consistent with basal cell markers including glutathione S-transferase-π and Bcl-2. This expression pattern was confirmed in human prostate cancer tissues. Our results also showed that prostate cancer cells positive with basal cell markers were more aggressive. Downregulation of Gal-3 expression resulted in increased apoptotic potential and decreased metastasis potential of prostate cancer cells. Our findings demonstrate for the first time that Gal-3 may serve as a new marker for basal characteristics of prostate cancer epithelium. This study helps us to better understand the heterogeneity of prostate cancer. The clinical significance of this study lies in the application of Gal-3 to distinguish prostate cancer subtypes and improve treatment efficacy with designed personalized therapy.

Galectin-3 regulates p21 stability in human prostate cancer cells.

Galectin-3 (Gal-3) is a multifunctional protein involved in cancer through regulation of cell adhesion, cell growth, apoptosis and metastasis, while p21 (Cip1/WAF1) is a negative regulator of the cell cycle, involved in apoptosis, transcription, DNA repair and metastasis. The results presented here demonstrate for the first time that the level of Gal-3 protein is associated with the level of p21 protein expression in human prostate cancer cells and the effects of Gal-3 on cell growth and apoptosis were reversed by modulating p21 expression level. Furthermore, Gal-3 regulates p21 expression at the post-translational level by stabilizing p21 protein via the carbohydrate-recognition domain. This is the first report suggesting a molecular function not yet described for Gal-3 as the regulator of p21 protein stability. This study provides a unique insight into the relationship of these two molecules during prostate cancer progression, and may provide a novel therapeutic target.

[Results of a novel real-time PCR, sequence analysis, Inno-LiPA line probe assays in the detection of hepatitis B virus G1896A precore mutation in French blood donors]. / Résultats de trois méthodes pour la détection de la mutation précore G1896A du virus de l'hépatite B chez les donneurs de sang français : PCR temps réel, séquençage et test Inno-LIPA.

AIM: To screen hepatitis B virus (HBV) genotypes and associated basal core promoter (BCP; T1762A/A1764) and precore (PC; A1896) mutations among the 100 HBV surface antigen (HBsAg) positive voluntary blood donors in France. METHODS: HBV genotypes were determined by using direct sequence analysis. Three methods were used to detect G1896A mutation: non-commercial real-time PCR (PCRTR°, line probe assay (InnoLiPA HBV PreCore, INNOGENETICS(®)) and direct sequencing of precore gene. HBV viral load was quantified with two commercial real-time PCR (COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV Test/Roche and Real Time HBV/M2000/Abbott). RESULTS: The mean age of donors was 30 (18-64). Patients were from Africa (42%), Europa (50%), and Asia (8%). HBV/D was the most predominant (37%) genotype followed by HBV/A (31%) and HBV/E (22%). PC and BCP mutants were found in 57% with Inno-LIPA HBV test and 59% with both PCRTR and sequencing methods. A significant difference in the viral load of blood donors with wild and PC mutants was observed with the Taqman Cobas real time PCR (3,19 Log(10) UI/ml versus 4,93 Log(10) UI/ml, p < 0.05). Precore phenotype determination was in agreement with the three PC mutation detection methods in 56% of cases. CONCLUSIONS: Non-Caucasian genotype E was present in the French blood donors. PC mutation was more common than BCP mutations in this study. As HBV infected blood donors were more often asymptomatic carriers, we could speculate that the G1896A mutation may favour the asymptomatic state, supporting previous observations.

Calpain activation through galectin-3 inhibition sensitizes prostate cancer cells to cisplatin treatment.

Prostate cancer will develop chemoresistance following a period of chemotherapy. This is due, in part, to the acquisition of antiapoptotic properties by the cancer cells and, therefore, development of novel strategies for treatment is of critical need. Here, we attempt to clarify the role of the antiapoptotic molecule galectin-3 in prostate cancer cells using siRNA and antagonist approaches. The data showed that Gal-3 inhibition by siRNA or its antagonist GCS-100/modified citrus pectin (MCP) increased cisplatin-induced apoptosis of PC3 cells. Recent studies have indicated that cisplatin-induced apoptosis may be mediated by calpain, a calcium-dependent protease, as its activation leads to cleavage of androgen receptor into an androgen-independent isoform in prostate cancer cells. Thus, we examined whether calpain activation is associated with the Gal-3 function of regulating apoptosis. Here, we report that Gal-3 inhibition by siRNA or GCS-100/MCP enhances calpain activation, whereas Gal-3 overexpression inhibits it. Inhibition of calpain using its inhibitor and/or siRNA attenuated the proapoptotic effect of Gal-3 inhibition, suggesting that calpain activation may be a novel mechanism for the proapoptotic effect of Gal-3 inhibition. Thus, a paradigm shift for treating prostate cancer is suggested whereby a combination of a non-toxic anti-Gal-3 drug together with a toxic chemotherapeutic agent could serve as a novel therapeutic modality for chemoresistant prostate cancers.

Kluyvera co-infection in two solid organ transplant recipients: an emerging pathogen or a colonizer bystander?

Kluyvera species are opportunistic, gram-negative bacilli in the family Enterobacteriaceae. Ordinarily occurring as a commensal, Kluyvera have been reported to cause serious infections in immunosuppressed and immunocompetent hosts, causing diarrhea, urinary infections, peritonitis, and cholecystitis. We report Kluyvera infections in 2 solid organ transplant recipients. An 18-year-old female with alpha-1 antitrypsin deficiency underwent living donor liver transplantation and presented 6 months later with a liver abscess. The abscess aspirate grew mixed organisms including Kluyvera cryocrescens. A 22-year-old female with renal failure secondary to focal segmental glomerulosclerosis underwent a deceased donor kidney transplant and presented 3 months later with pyelonephritis; the urine culture grew Kluyvera ascorbata. Both patients improved only when their antibiotic coverage was broadened to include Kluyvera. The isolation of Kluyvera as a pathogen in transplant patients emphasizes that this commensal organism may be virulent in this patient population.

Reduced priority MELD score for hepatocellular carcinoma does not adversely impact candidate survival awaiting liver transplantation.

The liver organ allocation policy of the United Network for Organ Sharing (UNOS) is based on the model for end-stage liver disease (MELD). The policy provides additional priority for candidates with hepatocellular carcinoma (HCC) who are awaiting deceased donor liver transplantation (DDLT). However, this priority was reduced on February 27, 2003 to a MELD of 20 for stage T1 and of 24 for stage T2 HCC. The aim of this study was to determine the impact of reduced priority on HCC candidate survival while on the waiting list. The UNOS database was reviewed for all HCC candidates listed after February 27, 2002, The HCC candidates were grouped into two time periods: MELD 1 (listed between February 27, 2002, and February 26, 2003) and MELD 2 (listed between February 27, 2003 and February 26, 2004). For the two time periods, the national DDLT incidence rates for HCC patients were 1.44 versus 1.53 DDLT per person-year (p = NS) and the waiting times were similar for the two periods (138.0 +/- 196.8 vs. 129.0 +/- 133.8 days; p = NS). Furthermore, the 3-, 6- and 12-month candidate, patient survival and dropout rates were also similar nationally. Regional differences in rates of DDLT for HCC were observed during both MELD periods. Consequently, the reduced MELD score for stage T1 and T2 HCC candidates awaiting DDLT has not had an impact nationally either on their survival on the waiting list or on their ability to obtain a liver transplant within a reasonable time frame. However, regional variations point to the need for reform in how organs are allocated for HCC at the regional level.

Binding and activation of thiamin diphosphate in acetohydroxyacid synthase.

Acetohydroxyacid synthases (AHASs) are biosynthetic thiamin diphosphate- (ThDP) and FAD-dependent enzymes. They are homologous to pyruvate oxidase and other members of a family of ThDP-dependent enzymes which catalyze reactions in which the first step is decarboxylation of a 2-ketoacid. AHAS catalyzes the condensation of the 2-carbon moiety, derived from the decarboxylation of pyruvate, with a second 2-ketoacid, to form acetolactate or acetohydroxybutyrate. A structural model for AHAS isozyme II (AHAS II) from Escherichia coli has been constructed on the basis of its homology with pyruvate oxidase from Lactobacillus plantarum (LpPOX). We describe here experiments which further test the model, and test whether the binding and activation of ThDP in AHAS involve the same structural elements and mechanism identified for homologous enzymes. Interaction of a conserved glutamate with the N1' of the ThDP aminopyrimidine moiety is involved in activation of the cofactor for proton exchange in several ThDP-dependent enzymes. In accord with this, the analogue N3'-pyridyl thiamin diphosphate does not support AHAS activity. Mutagenesis of Glu47, the putative conserved glutamate, decreases the rate of proton exchange at C-2 of bound ThDP by nearly 2 orders of magnitude and decreases the turnover rate for the mutants by about 10-fold. Mutant E47A also has altered substrate specificity, pH dependence, and other changes in properties. Mutagenesis of Asp428, presumed on the basis of the model to be the crucial carboxylate ligand to Mg(2+) in the "ThDP motif", leads to a decrease in the affinity of AHAS II for Mg(2+). While mutant D428N shows ThDP affinity close to that of the wild-type on saturation with Mg(2+), D428E has a decreased affinity for ThDP. These mutations also lead to dependence of the enzyme on K(+). These experiments demonstrate that AHAS binds and activates ThDP in the same way as do pyruvate decarboxylase, transketolase, and other ThDP-dependent enzymes. The biosynthetic activity of AHAS also involves many other factors beyond the binding and deprotonation of ThDP; changes in the ligands to ThDP can have interesting and unexpected effects on the reaction.

A nosocomial outbreak of fluoroquinolone-resistant salmonella infection.

BACKGROUND: Infection with fluoroquinolone-resistant strains of salmonella is rare, as is nosocomial salmonella infection. We describe the first recognized outbreak of fluoroquinolone-resistant salmonella infections in the United States, which occurred in two nursing homes and one hospital in Oregon. METHODS: We interviewed medical staff and reviewed patients' charts and death certificates. In Nursing Home A we conducted a case-control study. Patients were defined as residents of the nursing home from whom fluoroquinolone-resistant Salmonella enterica serotype Schwarzengrund was isolated between February 1996 and December 1998. Controls were residents with similar medical conditions whose cultures did not yield salmonella. We compared isolates using pulsed-field gel electrophoresis and sequence analysis. We reviewed pharmacy records to compare the use of fluoroquinolone among several nursing homes. RESULTS: Eleven patients with fluoroquinolone-resistant salmonellosis were identified at two nursing homes. The index patient had been hospitalized in the Philippines and had probably acquired the infection there. Transmission was probably direct (from patient to patient) or through contact with contaminated surfaces. Treatment with fluoroquinolones during the six months before a culture was obtained was associated with a significant risk of salmonella infection (4 of 5 patients had taken fluoroquinolones, as compared with 2 of 13 controls; odds ratio, 22.0; 95 percent confidence interval, 1.06 to 1177). The patients were not significantly more likely than the controls to have taken other antibiotics. More fluoroquinolones were used at Nursing Home A than at similar nursing homes in Oregon. The isolates from the outbreak had similar patterns on pulsed-field gel electrophoresis and the same gyrA mutations. The isolates from the outbreak were also similar to the only previous isolate of fluoroquinolone-resistant salmonella in the United States, which came from a patient in New York who had been transferred from a hospital in the Philippines. CONCLUSIONS: We describe a prolonged nosocomial outbreak of infection with fluoroquinolone-resistant S. enterica serotype Schwarzengrund. More such outbreaks are likely in institutional settings, particularly those in which there is heavy use of antimicrobial agents.

Rab3D, a small GTP-binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes.

Rab3 isotypes are expressed in regulated secretory cells. Here, we report that rab3D is also expressed in rat hepatocytes, classic models for constitutive secretion. Using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes. Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a protein of approximately 25 kd in hepatocyte lysates, hepatic subcellular fractions, and tissue extracts. The distribution of rab3D was primarily cytosolic; however, only membrane-associated rab3D significantly bound guanosine triphosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was enriched in a crude vesicle carrier fraction (CVCF), which includes transcytotic carriers. Vesicular compartments immunoisolated from the CVCF on magnetic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi marker protein. Second, indirect immunofluorescence on frozen liver sections and in polarized cultured hepatocytes localized rab3D-positive sites at or near the apical plasma membrane and to the pericanalicular cytoplasm. Finally, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytoplasm of cholestatic hepatocytes. Our results indicate that rab3D plays a role in the regulation of apically directed transcytosis in rat hepatocytes.

Autoimmune liver diseases. Recurrence after liver transplantation.

PBC and AIH recur after OLTx. The recurrence of PSC is less clear. Recurrence of these diseases seems to be of relatively little importance in the short term; however, longer follow-up is required to address the significance of recurrent disease. Immunosuppression in these patients may alter the natural history of these entities in the post-transplant setting.