The long noncoding RNA MALAT1 suppresses miR-211 to confer protection from ultraviolet-mediated DNA damage in vitiligo epidermis by upregulating sirtuin 1.

BACKGROUND: The absence of melanocytes poses a challenge for long-term tissue homeostasis in vitiligo. Surprisingly, while individuals with Fitzpatrick phototypes I-II (low melanin content) have a higher incidence of melanoma and nonmelanoma skin cancer, people with vitiligo are at a decreased risk for the same. OBJECTIVES: To understand the molecular mechanisms that protect vitiligo skin from ultraviolet (UV)-induced DNA damage by (i) characterizing differentially expressed microRNAs in lesional vs. nonlesional epidermis and (ii) identifying their upstream regulators and downstream gene targets. METHODS: Genome-wide microRNA profiling of nonlesional and lesional epidermis was performed on five individuals with stable nonsegmental vitiligo using next-generation RNA sequencing. The relevance of the upstream regulator and downstream target gene of the most differentially expressed microRNA was studied. RESULTS: Our study found sirtuin1 (SIRT1), an NAD-dependent deacetylase, to be a direct target of miR-211 - the most significantly downregulated microRNA in lesional epidermis. Inhibition of SIRT1 with EX-527 downregulated keratin 10 and involucrin, suggesting that SIRT1 promotes keratinocyte differentiation. Overexpression of miR-211 mimic led to a significant increase in γ-H2AX positivity and cyclobutane pyrimidine dimer (CPD) formation, hallmarks of UVB-mediated DNA damage. These effects could be ameliorated by the addition of resveratrol, a SIRT1 activator. Furthermore, a long noncoding RNA, MALAT1, was identified as a negative upstream regulator of miR-211. Overexpression of MALAT1 resulted in increased expression of SIRT1 and a concomitant removal of UVB-induced CPDs in primary keratinocytes. CONCLUSIONS: These findings establish a novel MALAT1-miR-211-SIRT1 signalling axis that potentially confers protection to the 'amelanotic' keratinocytes in vitiligo.

Poly(piperizinamide) with copper ion composite membranes: Application for mitigation of Hexaconazole from water and combat microbial contamination.

Thin film Poly(piperazine-amide) composite membranes using sequential interfacial polymerization with tuning by Cu2+ have brought significant findings in it. The hydrophobicity is relatively enhanced for the copper containing membranes. The membrane in which copper solution is applied prior to piperizine (Memb-III) exhibits higher hydrophobicity where as membrane (Memb-II) in which copper solution is applied following piperizine, possesses higher roughness compared to other two. Filtration experiments in terms of salts, mono/disaccharides and hexaconazole indicate that modified membranes are of different behaviours according to their sequence of preparative methods. Memb-III has shown lower SO4=/Cl- selectivity compared to Memb-II (i.e. 3.92), though they are in different range. The unmodified membrane (Memb-I) exhibits SO4=/Cl- selectivity 3.23 is in the same scale of Memb-III (2.27). Memb-III exhibits higher hexaconazole separation (91.5%) compared to Memb-II (i.e. 53.9%). The flux decline follows the order: field water > tap water > deionized water. The copper incorporated membrane (Memb-II) has shown a low flux decline compared to Memb-III as well as Memb-I. The antibacterial properties towards E. Coli and Bacillus subtilis are well reflected. The copper containing membranes have promising antibacterial properties and follows the order Memb-II > Memb-III > Memb-I.

Improvement of DNA recognition through molecular imprinting: hybrid oligomer imprinted polymeric nanoparticles (oligoMIP NPs).

High affinity and specific binding are cardinal properties of nucleic acids in relation to their biological function and their role in biotechnology. To this end, structural preorganization of oligonucleotides can significantly improve their binding performance, and numerous examples of this can be found in Nature as well as in artificial systems. Here we describe the production and characterization of hybrid DNA-polymer nanoparticles (oligoMIP NPs) as a system in which we have preorganized the oligonucleotide binding by molecular imprinting technology. Molecularly imprinted polymers (MIPs) are cost-effective "smart" polymeric materials capable of antibody-like detection, but characterized by superior robustness and the ability to work in extreme environmental conditions. Especially in the nanoparticle format, MIPs are dubbed as one of the most suitable alternatives to biological antibodies due to their selective molecular recognition properties, improved binding kinetics as well as size and dispersibility. Nonetheless, there have been very few attempts at DNA imprinting in the past due to structural complexity associated with these templates. By introducing modified thymine bases into the oligonucleotide sequences, which allow establishing covalent bonds between the DNA and the polymer, we demonstrate that such hybrid oligoMIP NPs specifically recognize their target DNA, and that the unique strategy of incorporating the complementary DNA strands as "preorganized selective monomers" improves the recognition properties without affecting the NPs physical properties such as size, shape or dispersibility.

Restoring expression of miR-16: a novel approach to therapy for malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is recalcitrant to treatment and new approaches to therapy are needed. Reduced expression of miR-15/16 in a range of cancer types has suggested a tumour suppressor function for these microRNAs, and re-expression has been shown to inhibit tumour cell proliferation. The miR-15/16 status in MPM is largely unknown. MATERIALS AND METHODS: MicroRNA expression was analysed by TaqMan-based RT-qPCR in MPM tumour specimens and cell lines. MicroRNA expression was restored in vitro using microRNA mimics, and effects on proliferation, drug sensitivity and target gene expression were assessed. Xenograft-bearing mice were treated with miR-16 mimic packaged in minicells targeted with epidermal growth factor receptor (EGFR)-specific antibodies. RESULTS: Expression of the miR-15 family was consistently downregulated in MPM tumour specimens and cell lines. A decrease of 4- to 22-fold was found when tumour specimens were compared with normal pleura. When MPM cell lines were compared with the normal mesothelial cell line MeT-5A, the downregulation of miR-15/16 was 2- to 10-fold. Using synthetic mimics to restore miR-15/16 expression led to growth inhibition in MPM cell lines but not in MeT-5A cells. Growth inhibition caused by miR-16 correlated with downregulation of target genes including Bcl-2 and CCND1, and miR-16 re-expression sensitised MPM cells to pemetrexed and gemcitabine. In xenograft-bearing nude mice, intravenous administration of miR-16 mimics packaged in minicells led to consistent and dose-dependent inhibition of MPM tumour growth. CONCLUSIONS: The miR-15/16 family is downregulated and has tumour suppressor function in MPM. Restoring miR-16 expression represents a novel therapeutic approach for MPM.

HIV and family planning service integration and voluntary HIV counselling and testing client composition in Ethiopia.

Integrating voluntary HIV counselling and testing (VCT) with family planning and other reproductive health services may be one effective strategy for expanding VCT service delivery in resource poor settings. Using 30,257 VCT client records with linked facility characteristics from Ethiopian non-governmental, non-profit, reproductive health clinics, we constructed multi-level logistic regression models to examine associations between HIV and family planning service integration modality and three outcomes: VCT client composition, client-initiated HIV testing and client HIV status. Associations between facility HIV and family planning integration level and the likelihood of VCT clients being atypical family planning client-types, versus older (at least 25 years old), ever-married women were assessed. Relative to facilities co-locating services in the same compound, those offering family planning and HIV services in the same rooms were 2-13 times more likely to serve atypical family planning client-types than older, ever-married women. Facilities where counsellors jointly offered HIV and family planning services and served many repeat family planning clients were significantly less likely to serve single clients relative to older, married women. Younger, single men and older, married women were most likely to self-initiate HIV testing (78.2 and 80.6% respectively), while the highest HIV prevalence was seen among older, married men and women (20.5 and 34.2% respectively). Compared with facilities offering co-located services, those integrating services at room- and counsellor-levels were 1.9-7.2 times more likely to serve clients initiating HIV testing. These health facilities attract both standard material and child health (MCH) clients, who are at high risk for HIV in these data, and young, single people to VCT. This analysis suggests that client types may be differentially attracted to these facilities depending on service integration modality and other facility-level characteristics.

Pentachlorophenol removal from water using surfactant-enhanced filtration through low-pressure thin film composite membranes.

Removal of pentachlorophenol from water is investigated using the surfactant-enhanced cross-flow membrane filtration technique in which anionic surfactant; sodium dodecyl sulfate (SDS) is the carrier of pentachlorophenol. The separation performances are studied by varying SDS concentrations (<

High-risk sexual behaviours among drug users in Pakistan: implications for prevention of STDs and HIV/AIDS.

Our objective was to describe HIV/STD risk behaviours and awareness among a community-based sample of drug users in Pakistan. Drug users contacted through street outreach by a non-governmental organization in Quetta, Peshawar and Rawalpindi underwent interviewer-administered questionnaires. Descriptive statistics were used to characterize sexual behaviours by city, marital status and the use of injection drugs. Logistic regression was used to identify correlates of ever having an STD. Of 608 drug users studied, all but one was male; median age was 32 years and 45% had no formal education. Half were married, of whom 25% were living with their wives. Sexual behaviours were reported as follows: 14% had sex with other males, 28% reported sex with males and females, 49% had paid money to have sex and only 10% had ever used condoms. One-fifth reported having had an STD and about 40% reported having suffered from either one or more STD-related symptoms. Only 41% had heard about HIV/AIDS, of whom 17% knew that HIV/AIDS could be transmitted through sexual contact. In conclusion, high-risk sexual behaviours are prevalent among male drug users in Pakistan, and awareness of transmission risks is low. These data attest to the urgent need for effective and specific interventions in Pakistan to prevent transmission of HIV and STDs among drug users and their sex partners (I J STD & AIDS 2004;15:601-7).

High-risk sexual behaviours among drug users in Pakistan: implications for prevention of STDs and HIV/AIDS.

Our objective was to describe HIV/STD risk behaviours and awareness among a community-based sample of drug users in Pakistan. Drug users contacted through street outreach by a non-governmental organization in Quetta, Peshawar and Rawalpindi underwent interviewer-administered questionnaires. Descriptive statistics were used to characterize sexual behaviours by city, marital status and the use of injection drugs. Logistic regression was used to identify correlates of ever having an STD. Of 608 drug users studied, all but one was male; median age was 32 years and 45% had no formal education. Half were married, of whom 25% were living with their wives. Sexual behaviours were reported as follows: 14% had sex with other males, 28% reported sex with both males and females, 49% had paid money to have sex and only 10% had ever used condoms. One-fifth reported having had an STD and about 40% reported having suffered from either one or more STD-related symptoms. Only 41% had heard about HIV/AIDS, of whom 17% knew that HIV/AIDS could be transmitted through sexual contact. In conclusion, high-risk sexual behaviours are prevalent among male drug users in Pakistan, and awareness of transmission risks is low. These data attest to the urgent need for effective and specific interventions in Pakistan to prevent transmission of HIV and STDs among drug users and their sex partners.

Short tandem repeat methodology for genotypic identification of single-person versus multi-person use of syringes.

OBJECTIVE: To develop laboratory methods to differentiate between single- versus multi-person use of syringes by injection drug users. METHODS: Forensic short tandem repeat (STR) genetic analysis was undertaken to cross-validate a test panel of trace blood contents from syringes representing single- versus multi-person syringe use. Laboratory-simulated scenarios of needle sharing generated 34 syringe washes that were blinded for evaluation. Polymerase chain reaction was used to amplify the polymorphic STR locus D6S502 from blood trace contents in used syringes. Alleles were sized and quantified using a commercial gene sequencer. A statistical algorithm was developed to determine the number of alleles present in the amplified DNA fragments. Syringes with more than two expected alleles were considered to represent multi-person syringe use. Sensitivity, specificity and the kappa coefficient were calculated. RESULTS: Allelic matrix-based analysis of alleles from the single STR successfully characterized single-use (n = 12) and multiple-use (n = 22) syringes with 68% sensitivity and 100% specificity upon re-analysis. The extent of agreement over and above chance (kappa = 0.6; P < 0.0001) indicated good agreement for differentiating single- versus multi-person syringe use. CONCLUSIONS: These findings suggest that improved genotypic STR analysis of syringe material could be an adjunct to methods for validating self-reported needle sharing, conducting behavioral surveillance of needle-sharing behaviors, and evaluating interventions such as needle-exchange programs. Assays based on multiple STR loci will undoubtedly improve upon the promising results obtained from laboratory simulations of needle sharing.

Characteristics and utilization patterns of needle-exchange attendees in Chicago: 1994-1998.

The objectives of this study were to describe characteristics and utilization patterns of participants attending a needle-exchange program (NEP) in Chicago, Illinois. Since 1994, demographics of NEP attendees and program utilization data were collected systematically at 22 sites operated by the Chicago Recovery Alliance (CRA). Descriptive statistics were used to assess time trends, site variations, and characteristics of attendees in day sites versus evening sites. A total of 11,855 injection drug users (IDUs) visited the NEP at least once from 1994 to 1998. Median age was 41 years, and 74% were male. Overall race distribution was African-American 50%, Caucasian 38%, Puerto Rican 10%, other 2%. Over time, there was a proportional decrease in African-American users (55.4% to 39.9%, P < .001), a significant increase in Puerto Rican users (1.4% to 14.1%, P < .001), and a stable proportion of Caucasian users (42%). Each year, 15-20% of all clients were first-time attenders. Overall, participants attending evening versus day sites were younger (median age 39 years vs. 42 years, P < .001) and more ethnically diverse. Over a 4-year period, this NEP reached a diverse population of IDUs and recruited a stable proportion of first-time users. Compared to daytime NEP venues, evening NEP sites attracted younger clients and those who were more diverse ethnically. To maximize coverage of sterile syringes, NEPs should strive for diversification in terms of hours of operation and location.

Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glial cortical cultures.

Nitric oxide (NO) is a potent biological messenger molecule in the central nervous system (CNS). There are several potential sources of NO production in the CNS, including neurons and endothelial cells which express NO synthase (NOS) constitutively. Astrocytes and microglia can be induced by cytokines to express a NOS isoform similar to macrophage NOS (mNOS). Primary mixed glial cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and gamma-interferon (INF-gamma) produce nitrite, a breakdown product of NO formation, in a dose-dependent manner. Nitrite production is detectable at 12 hr, peaks at 48 hr and is sustained for at least 96 hr. The NOS inhibitor, nitro-L-arginine (NArg), inhibits nitrite formation, but the immunosuppressant agent, FK506, does not. In mixed glial-neuronal cultures exposed to 50 ng LPS or 5 ng LPS and 1 microgram INF-gamma, neurons begin to die at 48 hr, approx. 24-36 hr after detectable nitrite production. Neurotoxicity is attenuated by 100 microM NArg. These data indicate that expression of inducible mNOS causes delayed neurotoxicity.

Expression of Shiga toxin B subunit at cell surface in E. coli K-12.

The three parts (Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the LamB fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.

Stimulation of secretory antibodies against Bordetella pertussis antigens in the lungs of mice after oral or intranasal administration of liposome-incorporated cell-surface antigens.

Lipopolysaccharide (LPS) and outer membrane protein (OMP) preparations of Bordetella pertussis were incorporated into multilamellar liposomes composed of soya bean-derived phospholipids which were then used for oral and intranasal immunization of mice. Specific antibody responses of animals immunized by either route were measured in lung washes. A specific IgA response to LPS was detected after immunization with the OMP-containing liposomes but not with the LPS-containing liposomes, indicating adjuvant activity of the proteins. The OMP-containing liposomes were significantly more effective in inducing immune responses than the OMP preparation alone. Responses were highest when mice were given a booster 30 days after primary immunization. Maximum responses occurred 20 days after the booster but specific antibody was still detected 75 days after the secondary immunization. These results suggest that this liposome antigen delivery system has potential in stimulating secretory antibody responses which may be helpful in protecting against infection from B. pertussis.

[Secretive export of StxB/HlyA (CT) fusion protein and B subunit specific antibody responses in mice].

In this study, the Shiga toxin B subunit has been fused to haemolysin A C-terminus. The fusion protein StxB/HlyA(CT) can be exported to the external medium not only from E. coli K-12 but also from S. typhimurium aroA strain SL3261. When the plasmid pUC18 was used as vector, StxB/HlyA(CT) was toxic to hosts. But the fusion protein was stable and safe to hosts when the pUC18 was replaced with pBR322. The fusion protein can be expressed and exported to the external medium under either the aerobactin promoter or beta-lactomase promoter. Oral and i.p. immunization of mice with StxB/HlyA(CT) carrying S. typhimurium aroA strain SL3261 resulted in significant B subunit specific mucosal and serum antibody responses. This the first report demonstrating that foreign polypeptides fused to the 23kD C-terminus of E. coli haemolysin A can be exported from attenuated Salmonella Vaccine strains and that such exported polypeptides can result in antigen specific immune responses.

Extracellular export of Shiga toxin B-subunit/haemolysin A (C-terminus) fusion protein expressed in Salmonella typhimurium aroA-mutant and stimulation of B-subunit specific antibody responses in mice.

The Shiga toxin B-subunit has been fused to the 23-kD C-terminus of Escherichia coli haemolysin A (HlyA) and exported from attenuated antigen carrier strain of Salmonella typhimurium aroA (SL3261). The expression of the gene fusion under the control of a synthetic modified beta-lactamase promoter (constitutive expression) and under the iron-regulated aerobactin promoter showed that the fusion protein could be stably expressed and exported out of the bacterial cell in significant amounts so long as high copy number plasmids were not used. Oral and i.p. immunization of mice with the hybrid salmonellae resulted in significant B-subunit specific mucosal and serum antibody responses. A comparative analysis of the location of hybrid proteins in the antigen carrier bacterial cell (i.e. cytoplasmic expression and extracellular export) has shown that both modes of expression result in antigen-specific immune responses. This is the first report demonstrating that foreign polypeptides fused to the 23-kD C-terminus of E. coli haemolysin A can be exported from attenuated Salmonella vaccine strains and that such exported polypeptides can result in antigen-specific immune responses.

Construction of stable LamB-Shiga toxin B subunit hybrids: analysis of expression in Salmonella typhimurium aroA strains and stimulation of B subunit-specific mucosal and serum antibody responses.

The complete Shiga toxin B subunit and two N-terminal segments of the B subunit have been inserted into a cell surface exposed loop of the LamB protein, and expression of the hybrid proteins from three different promoter systems, i.e., (i) an in vitro-inducible tac promoter that provides high-level expression, (ii) the iron-regulated aerobactin promoter presumably induced in vivo under the iron-limiting conditions of the intestinal mucosal environment, and (iii) a synthetic, modified beta-lactamase promoter providing moderate level constitutive expression, has been analyzed in Escherichia coli, Salmonella typhimurium, and attenuated antigen carrier strains of S. typhimurium (aroA mutants). The hybrid vaccine strains were used to immunize mice by the oral and intraperitoneal routes. S. typhimurium aroA mutants apparently have a membrane export defect which prevents the transport of LamB and its derivatives across the cytoplasmic membrane. High-level expression of hybrid proteins through use of the tac promoter proved deleterious to the vaccine strains and prevented the production of viable cells at reasonable cell densities. The lower levels of gene expression observed with the beta-lactamase and aerobactin promoters did not have this effect. Immunization of mice with S. typhimurium aroA strains carrying the hybrid genes expressed from these two promoters resulted in significant B subunit-specific mucosal and serum antibody responses. This suggests that such expression systems may be useful when incorporated into candidate antidysentery live oral vaccines for inducing protection against the effect of Shiga toxin in infections caused by Shigella dysenteriae 1 and other Shiga toxin-or Shiga-like toxin-producing pathogens.

Complete physical map of the rfb gene cluster encoding biosynthetic enzymes for the O antigen of Salmonella typhimurium LT2.

Biosynthesis of the Salmonella typhimurium LT2 O antigen is encoded by genes which map in the rfb cluster. The cloning and restriction enzyme analysis of part of this cluster have been described previously (H. N. Brahmbhatt, N. B. Quigley, and P. R. Reeves, Mol. Gen. Genet. 203:172-176, 1986). The entire rfb gene cluster has now been cloned, and a detailed restriction enzyme map has been constructed which has enabled us to map the approximate positions of individual rfb genes.

A low copy number cosmid.

A low copy number cosmid was constructed by subcloning the pair of cos sites and the kanamycin resistance gene of pcos2EMBL into pGB2. The resulting cosmid, pPR691, has the pSC101 replicon and specifies resistance to kanamycin, spectinomycin, and streptomycin. pPR691 also carries restriction sites suitable for cloning partial Sau3A digests using the strategy of Bates and Swift (P. F. Bates and R. A. Swift, 1983, Gene 26, 137-146). A library of Salmonella typhimurium chromosomal DNA was made using this cosmid and the rfb gene cluster (map position 42) was isolated from this library.