Feasibility and impact of a one-stop thyroid clinic in a low- and middle-income country.

BACKGROUND: The study was done to evaluate the feasibility, safety and outcomes of a one-stop thyroid clinic (OSTC) in a low- and middle-income country (LMIC) setting. METHODS: This was a prospective non-randomised case control study consisting of patients with thyroid nodules evaluated and managed at a tertiary referral centre in an LMIC between February 2019 and January 2020. Patients were divided into two groups based on the kind of preoperative evaluation protocol: OSTC group (n = 118) - OSTC protocol, and control group (CG, n = 108) - routine protocol. RESULTS: Baseline clinical characteristics of the two groups including median age (p = 0.13) and gender distribution (p = 0.76) were comparable. The majority of patients in both groups belonged to a low-income group (46.6% vs 47.3%; p = 0.91), followed by a middle-income group (35.6% vs 30.5%; p = 0.41). The median number of outpatient department visits (1 vs 3 days; p = < 0.001), waiting time for neck ultrasonography (1 vs 3 days; p = < 0.0001), fine needle aspiration cytology (1 vs 2 days; p = < 0.0001), and out of pocket expenditure (INR 3 965 vs 6 624; p = < 0.001) was significantly less in the OSTC group compared to the CG. Patients in the OSTC group reported better satisfaction levels (p = < 0.0001). Accuracy of diagnosis did not differ between the two groups (p = 0.14). CONCLUSION: OSTC practice is feasible, provides comparative clinical outcomes and seems cost effective in an LMIC. This protocol can be adopted as a routine practice in any health system.

Endodontic management of badly broken down teeth using the canal projection system: two case reports.

AIM: Teeth that have been weakened by caries and require root canal treatment to maintain their functional integrity may present with minimal coronal tooth structure and are a challenge for isolation and restoration. The aim of this clinical report is to demonstrate the management of badly broken down teeth using the Projector Endodontic Instrument Guidance System (PEIGS). SUMMARY: The PEIGS is an adjunct to root canal treatment designed to enhance the ease of treatment delivery. Use of this system facilitates projection of canal orifices from the floor of the pulp chamber to the cavosurface, providing direct visualization of and physical access to the projected canals. This report demonstrates the use of this novel device for the management of two badly broken down teeth. KEY LEARNING POINTS: Use of the endodontic projection system has the following advantages: * 'Projects' the canal orifice from the floor of the pulp chamber to the cavosurface, thereby enhancing visualization and access to the canals. * The bonded coronal build up reduces the risk of interappointment crack initiation and coronal-radicular fracture of weakened tooth structure. * Permits individualization of canals especially when they lie in close proximity to each other on the chamber floor. * Isolation may be facilitated by ease of clamp retention, rendering many structurally debilitated teeth endodontically treatable.

Conditional transformation of rat embryo fibroblast cells by a cyclin D1-cdk4 fusion gene.

Cyclin D1 gene overexpression is a frequent event in a number of human cancers. These observations have led to the suggestion that cyclin D1 alterations might play a role in the etiology of cancer. This possibility is supported by the finding that transfection of mammalian cells with cyclin D1 can accelerate progression through the G1 phase of the cell cycle. Moreover, cyclin D1 can function as an oncogene by cooperating with activated Ha-ras to transform primary rat embryo fibroblasts (REFs). In addition, cyclin D1 transgenics develop hyperplasia and neoplasia of the thymus and mammary gland. We have constructed a novel fusion gene consisting of full-length human cyclin D1 and cdk4 genes. This fusion gene was expressed in insect cells and the fusion protein was shown to be enzymatically active. The fusion gene was expressed in mammalian cells under the control of tet-repressor. This fusion gene immortalized primary REFs, and cooperated with activated Ha-ras to transform primary REFs, in terms of anchorage-independent growth in vitro and formation of tumors in vivo. Utilizing a tet-regulated gene expression system, we have shown that proliferation of stably transfected primary REFs in vitro and in vivo is dependent on the continued expression of the cyclin D1-cdk4 fusion gene. These cell lines could be useful in the discovery of novel cancer therapeutics to modulate cyclin D1.cdk4 activity.

Analysis of the integration function of the streptomycete bacteriophage phi C31.

A 2.1 kb (1 kb = 10(3) base-pairs) segment of DNA from the streptomycete bacteriophage phi C31 was found to be sufficient to direct site-specific integration of plasmid vectors in Streptomyces ambofaciens and Streptomyces fradiae in the absence of any streptomycete origin of replication. Sequencing and analysis of phage, chromosomal and junction attachment sites of S. ambofaciens and S. fradiae revealed that recombination is conservative and that crossover takes place within three bases of homology between phage and host. Deletion analysis, sequencing and site-specific mutagenesis of the phi C31 DNA revealed a large open reading frame (ORF 613) whose expression was necessary for integration. This ORF begins near the point of crossover and reads away from the attachment site. A comparison of the predicted amino acid sequence of ORF 613 with known recombinases did not reveal any significant similarities. A genetic analysis of the amino-terminal region of ORF 613 suggested that translation could initiate at any one of three possible start codons. Primer extension experiments showed that transcriptional initiation occurred at a T and a C only four and five bases, respectively, from the site of crossover. This analysis suggested that ORF 613 would be separated from its promoter upon integration.

Chronic renal insufficiency from cortical necrosis induced by arsenic poisoning.

A 39-year-old man had anuria and azotemia and was found to be suffering from acute arsenic poisoning. After two peritoneal dialyses, partial renal function returned, and the patient has survived for five years without dialysis. Renal cortical necrosis was demonstrated by renal biopsy and renal calcification. We suggest that arsenic be added to the list of substances capable of causing renal cortical necrosis and recommend consideration of this complication in cases of arsenical poisoning.

Construction and properties of plasmid pKC30, a pBR322 derivative containing the pL-N region of phage lambda.

The phage lambda N gene, contained on a HindIII-BamHI lambda DNA fragment (lambda coordinates 38 695-34 500), was inserted into plasmid pBR322 and cloned in Escherichia coli containing a defective lambda prophage. The plasmid is called pKC30. lambda N-mediated pL transcription interfered with plasmid pKC30 maintenance. Plasmid pKC30 was stabilized (i) by repression of pL, (ii) by a nutL mutation, or (iii) by cloning an "N-unresponsive terminator" sequence downstream from N gene.

A thiostrepton-inducible expression vector for use in Streptomyces spp.

A shuttle expression vector containing the thiostrepton-inducible Streptomyces lividans promoter, ptipA, and the origin of transfer from plasmid RP4 was constructed. Cassettes containing a promoterless xylE gene upstream from a hyg gene were used to demonstrate thiostrepton-inducible expression from ptipA in both S. lividans and Streptomyces ambofaciens, ptipA was estimated to be induced 60-fold or more in Streptomyces ambofaciens.

Cloning a replication function from the streptomycete bacteriophage FP43.

Streptomyces griseofuscus cells carrying a 4.4-kb SphI DNA fragment from bacteriophage FP43 inhibited plaque formation (Pin) by FP43, and the Pin function was localized to a 0.96-kb SacII fragment. The same 4.4-kb SphI fragment was able to replicate freely in several streptomycetes, including S. griseofuscus, and the replication (Rep) function was localized to a 1.2-kb SphI-FspI fragment. Plasmids with FP43 Rep function are unstable and are present at about 20-50 copies per chromosome. Plasmids with FP43 Rep function are compatible with SCP2* plasmids.

Expression in Streptomyces ambofaciens of an Escherichia coli K-12 gene which confers resistance to hygromycin B.

We have constructed two plasmid vectors (pKC293 and pKC305) that can replicate in Escherichia coli K-12 and Streptomyces ambofaciens. These shuttle vectors were used to demonstrate the expression of two E. coli genes, hygromycin B (Hm) resistance and Tn5 neomycin (Nm) resistance, in S. ambofaciens.

Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA segments.

Plasmid pKC7, a derivative of pBR322, specifies resistance to both ampicillin and kanamycin. The DNA of this small plasmid (5.8 kb) contains unique sites for insertion of DNA cleaved with ten different restriction endonucleases. A detailed restriction endonuclease cleavage map is presented. The utility of this plasmid for cloning is discussed.

A thermoinducible lambda phage-ColE1 plasmid chimera for the overproduction of gene products from cloned DNA segments.

Two segments of lambda have been cloned into the multicopy plasmid pBR322. One extends from N through cII (NcII segment, from 71.3 to 81.0% on the physical map) and the other from N through P (NOP segment, from 71.3 to 86.5% on the physical map). Cells carrying these recombinant plasmids express lambda immunity (cIts) and Rex function. In addition, they decrease the efficiency of plating at 32 degrees C of lambdavir and lambdaimm434, but not that of lambdaimm21. Recombinant plasmids with lambdaNOP segments (pKC14, pKC16) differ from recombinant plasmid with labmdaNcII segment (pKC10) in two respects: (i) strains carrying pKC14 or pKC16 are killed at 42 degrees C, and (ii) these strains are thermally inducible for plasmid DNA synthesis, resulting in increase of plasmid copy number from an uninduced level of 50 to more than 130 per chromosome. It was suggested that both these differences are related to functions contained in the lambda DNA segment extending from 81.0 to 86.5%. The usefulness of plasmid pKC16 for overproduction of gene products from cloned DNA segments was demonstrated by cloning the E. coli exonuclease III gene (xth) in pKC16. Thermal induction of this xth plasmid (pSGr) results in a 125-fold increase in exonuclease III activity over that of a control strain lacking the xth gene insert. The extent of exonuclease III overproduction obtained by cloning xth gene in a lambda vector was similar to that obtained with pSGR3.

Plasmid cloning vectors that integrate site-specifically in Streptomyces spp.

Cloning vectors based on the Streptomyces ambofaciens plasmid pSAM2 and the streptomycete phage phi C31 were developed for use in Streptomyces spp. These vectors replicate in Escherichia coli but integrate by site-specific recombination in Streptomyces spp. Both pSAM2-based and phi C31-based vectors transformed a number of different Streptomyces spp; however, the phi C31-based vectors consistently transformed at higher frequencies than pSAM2-based vectors. Southern analysis indicated that the phi C31-based vectors integrated at a unique site in the S. ambofaciens chromosome, while the pSAM2-based vectors gave complex patterns which could indicate structural instability or use of multiple loci. Both types of vectors utilize the apramycin (Am)-resistance gene which can be selected in E. coli and Streptomyces spp. with either Am or the commercially available antibiotic Geneticin (G418).

Construction and characterization of a one-plasmid system for the controlled expression of genes in mammalian cells by tetracycline.

The two-plasmid system of Gossen and Bujard [Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551] to express mammalian genes in a tetracycline-repressed fashion was combined into a single-plasmid system. Two variants of this single-plasmid system that differ in the multiple cloning site (MCS) region are described. These vectors were used to stably transfect raf kinase domain into the normal rat kidney epithelial cell line (NRKE) to obtain a conditionally transformed cell line. These vectors were also used to stably transfect wild-type and mutant human p53 into the human osteosarcoma cell line, SAOS-2. Tetracycline repressed gene expression in both cell lines; about 12-fold in NRKE and about 80-fold in SAOS-2 cell line.

Cloning of restriction and modification genes in E. coli: the HbaII system from Haemophilus haemolyticus.

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R-PstI site, (formula: see text), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3'-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3'-OH termini, thus regenerating the PstI cleavage site sequences. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r-m-recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage lambdacI-0). A single phage-resistant clone was found. The recombinant plasmid, pD110, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.

Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for the conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.

A new shuttle cosmid vector, pKC505, for streptomycetes: its use in the cloning of three different spiramycin-resistance genes from a Streptomyces ambofaciens library.

A new shuttle cosmid vector, pKC505, was constructed for the cloning of Streptomyces DNA. This vector, which can be conjugally transferred between different streptomycetes, was used to construct a genomic library from a spiramycin-producing S. ambofaciens strain. By transformation of the spiramycin-sensitive S. griseofuscus with the library, three phenotypically different spiramycin-resistance genes were isolated. S. ambofaciens DNA in these clones was colinear with the chromosome, and the cloned DNA was stable in E. coli, S. griseofuscus and S. fradiae. These cosmids could be isolated easily from S. griseofuscus, an improvement over the previous shuttle cosmid vector, pKC462a [Stanzak et al., Bio/Technology 4 (1986) 229-232], which was somewhat difficult to isolate from S. lividans.

Production of a novel polyketide through the construction of a hybrid polyketide synthase.

The lactone rings of the polyketides platenolide and tylactone are synthesized by condensation of acetate-, proprionate-, and butyrate-derived precursors. A hybrid tylactone/platenolide synthase was constructed to determine if the choice of substrate is programmed by the polyketide synthase and to ascertain if a substrate different than that normally used in the first step of platenolide synthesis could be incorporated into the final polyketide. In this work, we report the successful incorporation of a propionate in place of the acetate normally used in the first step of platenolide synthesis. This result demonstrates that polyketide synthases choose a particular substrate at defined steps and provides strong evidence that substrate choice is programmed by the acyl transferase domain of a large, multifunctional polyketide synthase.

Expression of prokaryotic genes for hygromycin B and G418 resistance as dominant-selection markers in mouse L cells.

Novel bacterial resistance genes were cloned and expressed as dominant selection markers in mammalian cells. Escherichia coli genes coding for resistance to the aminocyclitol antibiotics hygromycin B (Hm) and G418 were cloned into the eukaryotic expression plasmid pSV5GPT [Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78 (1981) 2072-2076]. Mouse cells normally sensitive to 100 micrograms/ml Hm were transformed with these plasmids and selected in 200 micrograms/ml Hm. Transformants resistant to as much as 1 mg/ml Hm and 500 micrograms/ml G418 were isolated. Cell extracts contained an acetyltransferase activity capable of acetylating G418 and an Hm aminocyclitol phosphotransferase activity. Plasmid DNA sequences were identified by Southern blot analysis of high Mr DNA isolated from transformed cells.

Sequence similarity between macrolide-resistance determinants and ATP-binding transport proteins.

The three macrolide-resistance-encoding genes, tlrC from Streptomyces fradiae, srmB from Streptomyces ambofaciens, and carA from Streptomyces thermotolerans, encode proteins that possess significant sequence similarity to ATP-dependent transport proteins. The N-terminal and C-terminal halves of these proteins are very similar to each other and contain highly conserved regions that resemble ATP-binding domains typically present within the superfamily of ATP-dependent transport proteins. These observations suggest that the mechanism by which these genes confer resistance to macrolides is due to export of the antibiotics, a process that is driven by energy derived from ATP hydrolysis.