Estimation of the rate of tooth wear in permanent incisors: a cross-sectional digital radiographic study.

This study used conventional digital radiography to estimate the rate of tooth wear (TW) of maxillary and mandibular central incisors based on a cross-sectional study design. The crown length of 1239 permanent maxillary and mandibular central incisors from 346 persons (age groups: 10, 25, 40, 55 and 70 years ± 3) were measured by three calibrated dentists. Study teeth were intact incisally, had clearly visible incisal edges and cementoenamel junctions and had natural tooth antagonists. Measures were based on digital radiographic images (N = 666) archived in MiPACS within the electronic health record (axiUm(®)) from the College of Dentistry patient database. Incisor crown length decreased at a linear rate in both arches over the 60 years represented by the age groups. The average crown length for maxillary incisors in the youngest age group was 11.94 mm, which decreased by an average of 1.01 mm by median age 70. For mandibular incisors, the average crown length in the youngest age group was 9.58 mm, which decreased by an average of 1.46 mm in the oldest age group. Males and females showed similar rates of TW. Regardless of age, females demonstrated smaller mean crown height for maxillary incisors than males (P < 0.0001). Measures by the examiners demonstrated good agreement, with an interclass correlation coefficient of 0.869 and an average intra-examiner correlation of 99.5%, based on repeated measurements (n = 100). TW was estimated to average 1.01 mm for maxillary central incisors and 1.46 mm for mandibular central incisors by age 70 years.

A comparative study of psychosocial determinants and mental well-being in chronic kidney disease patients: A closer look.

BACKGROUND: Depressive illness in chronic kidney disease (CKD) is an independent risk factor for morbidity and mortality. The relation between depressive illness and quality of life (QoL) in this vulnerable group is complex. We attempted to study the burden of depressive illness, the QoL in patients of CKD on hemodialysis (HD), and peritoneal dialysis (PD) as well as those who were not on any dialysis but on conservative medical management only. MATERIALS AND METHODS: Observational study with cross-sectional analytical controlled design. STATISTICAL METHODS USED: Chi-square statistic or Fisher's exact test for categorical variables and t-test and ANOVA for continuous variables. Correlational analysis was done using Spearman's correlation coefficient. P <0.05 was considered as statistically significant. RESULTS: Depressive symptoms were present significantly across all 3 groups of CKD patients. Depressive disorder was significantly higher in the HD group. Mean QoL was significantly better in patients of CKD on PD. DISCUSSION: There is huge hidden burden of depressive symptoms and depressive illness in patients of CKD whether on dialysis or on conservative medical management. The study found significantly higher burden of depressive illness and lower QoL among the HD vis a vis PD patients, even though the majority of our CKD who require dialysis are on HD. CONCLUSION: Depressive burden is the hidden factor behind poor QoL, poor overall satisfaction as well as treatment outcome in patients of CKD whether or not on dialysis. To address this hidden depressive burden comprehensively, close cooperation between nephrologist and psychiatrist is called for.

Long-Term Consequences of Complex Living Renal Donation: Is It Safe?

INTRODUCTION: As there is a paucity of literature regarding the long-term outcomes of complex living donors, we conducted this study to assess the effect of kidney donation on the complex living kidney donor. MATERIALS AND METHODS: This retrospective study was conducted in Narayan Health Hospital, Kolkata, Eastern India. The cohort consisted of complex living kidney donors who donated kidneys between the years 2007 and 2012. All donors were 60 years old or older, or were younger than 60 years and had comorbidities like hypertension and obesity. After a minimum follow-up of 5 years, all donors underwent evaluation. Data pertaining to hypertension, new-onset diabetes, body mass index (BMI), estimated glomerular filtration rate (eGFR) and albuminuria, and cardiac events were compared from the time of donation till 5 years post-transplant. RESULTS AND DISCUSSION: We found a statistically significant increase in blood pressure, number of antihypertensives used, and mean BMI at follow-up. Diabetes mellitus was developed in 22.3% of donors. The mean GFR also decreased significantly at follow-up. There were 42 elderly donors (≥60 years) and 23 ≤ 59 years of age. There was a significant fall of eGFR in both groups, but the percentage fall was similar in both groups. A significant percentage of donors developed proteinuria, the majority being hypertensives. CONCLUSION: Procurement of kidneys from marginal donors should be done cautiously, and donors should be assessed for morbidity and mortality in the future, as we found a statistically significant deterioration in renal function, blood pressure, and BMI over long-term follow-up.

Conserved sequence blocks in kinetoplast minicircles from diverse species of trypanosomes.

Kinetoplast DNA minicircles from various species of trypanosomes are heterogeneous in nucleotide sequence to various degrees but in all instances contain a conserved sequence region of 100 to 200 base pairs present in one, two, or four copies per minicircle. Comparison of the conserved sequence regions of minicircles from eight species of trypanosomes revealed a common sequence motif consisting of three conserved sequence blocks (CSBs) present in the same order and with similar spacing in all species. In addition to the invariant 12-base-pair universal minicircle sequence (CSB-3), a 10-base-pair sequence (CSB-1) and an 8-base-pair sequence (CSB-2) are highly conserved in all minicircles. The overlap of CSB-1 and CSB-3 with previously identified 5' termini of newly synthesized minicircle H and L strands, respectively, and the presence of this conserved sequence motif in minicircles from diverse species suggest that these CSBs may determine a common mechanism of minicircle replication.

Replication of DNA minicircles in kinetoplasts isolated from Crithidia fasciculata: structure of nascent minicircles.

We have previously described an isolated kinetoplast system from Crithidia fasciculata capable of ATP-dependent replication of kinetoplast DNA minicircles (L. Birkenmeyer and D.S. Ray, J. Biol. Chem. 261: 2362-2368, 1986). We present here the identification of two new minicircle species observed in short pulse-labeling experiments in this system. The earliest labeled minicircle species (component A) contains both nascent H and L strands and is heterogeneous in sedimentation and electrophoretic migration. Component A has characteristics consistent with a Cairns-type structure in which the L strand is the leading strand and the H strand is the lagging strand. The other new species (component B) has a nascent 2.5-kilobase linear L strand with a single discontinuity that mapped to either of two alternative origins located 180 degrees apart on the minicircle map. Component B could be repaired to a covalently closed form by Escherichia coli polymerase I and T4 ligase but not by T4 polymerase and T4 ligase. Even though component B has a single gap in one strand, it had an electrophoretic mobility on an agarose gel (minus ethidium bromide) similar to that of a supercoiled circle with three supertwists. Treatment of component B with topoisomerase II converted it to a form that comigrated with a nicked open circular form (replicative form II). These results indicate that component B is a knotted topoisomer of a kinetoplast DNA minicircle with a single gap in the L strand.

Identification of cis and trans elements involved in the cell cycle regulation of multiple genes in Crithidia fasciculata.

Transcripts of several DNA replication genes, including the RPA1 and TOP2 genes, encoding the large subunit of nuclear replication protein A and the kinetoplast topoisomerase II, accumulate periodically during the cell cycle in the trypanosomatid Crithidia fasciculata. An octamer consensus sequence, CAUAGAAG, present in the 5' untranslated regions (UTR) of these mRNAs is required for periodic accumulation of the TOP2 and RPA1 transcripts and also for binding of a nuclear factor(s) to the 5' UTR RNAs of these genes. We show here that insertion of multiple (six) copies of this octamer sequence (6x octamer) into the 5' UTR of a reporter gene confers periodic accumulation on its transcript. Competition experiments and UV cross-linking studies show that the 6x octamer RNA and TOP2 5' UTR RNA bind to the same nuclear factor(s). Single-nucleotide substitutions in the 6x octamer that abolish the RNA gel shift also prevent cyclic accumulation of the reporter gene transcript. A protein termed cycling element binding protein, purified by affinity chromatography using 6x octamer RNA as a ligand, binds to RNAs containing wild-type octamers and not to those with mutant octamers. These results define a small sequence element in C. fasciculata mRNAs required for their cell cycle regulation and report the identification and purification of a putative regulatory protein that binds specifically to these elements.

Sequences within the 5' untranslated region regulate the levels of a kinetoplast DNA topoisomerase mRNA during the cell cycle.

Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA.

Characterization of the Crithidia fasciculata mRNA cycling sequence binding proteins.

The Crithidia fasciculata cycling sequence binding protein (CSBP) binds with high specificity to sequence elements in several mRNAs that accumulate periodically during the cell cycle. Mutations in these sequence elements abolish both cycling of the mRNA and binding of CSBP. Two genes, CSBPA and CSBPB, encoding putative subunits of CSBP have been cloned and were found to be present in tandem on the same DNA molecule and to be closely related. CSBPA and CSBPB are predicted to encode proteins with sizes of 35.6 and 42.0 kDa, respectively. Both CSBPA and CSBPB proteins have a predicted coiled-coil domain near the N terminus and a novel histidine and cysteine motif near the C terminus. The latter motif is conserved in other trypanosomatid species. Gel sieving chromatography and glycerol gradient sedimentation results indicate that CSBP has a molecular mass in excess of 200 kDa and an extended structure. Recombinant CSBPA and CSBPB also bind specifically to the cycling sequence and together can be reconstituted to give an RNA gel shift similar to that of purified CSBP. Proteins in cell extracts bind to an RNA probe containing six copies of the cycling sequence. The RNA-protein complexes contain both CSBPA and CSBPB, and the binding activity cycles in near synchrony with target mRNA levels. CSBPA and CSBPB mRNA and protein levels show little variation throughout the cell cycle, suggesting that additional factors are involved in the cyclic binding to the cycling sequence elements.

Nucleus-encoded histone H1-like proteins are associated with kinetoplast DNA in the trypanosomatid Crithidia fasciculata.

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomatids, consists of thousands of minicircles and 20 to 30 maxicircles catenated into a single large network and exists in the cell as a highly organized compact disc structure. To investigate the role of kinetoplast-associated proteins in organizing and condensing kDNA networks into this disc structure, we have cloned three genes encoding kinetoplast-associated proteins. The KAP2, KAP3, and KAP4 genes encode proteins p18, p17, and p16, respectively. These proteins are small basic proteins rich in lysine and alanine residues and contain 9-amino-acid cleavable presequences. Proteins p17 and p18 are closely related to each other, with 48% identical residues and carboxyl tails containing almost exclusively lysine, alanine, and serine or threonine residues. These proteins have been expressed as Met-His6-tagged recombinant proteins and purified by metal chelate chromatography. Each of the recombinant proteins is capable of compacting kDNA networks in vitro and was shown to bind preferentially to a specific fragment of minicircle DNA. Expression of each of these proteins in an Escherichia coli mutant lacking the HU protein rescued a defect in chromosome condensation and segregation in the mutant cells and restored a near-normal morphological appearance. Proteins p16, p17, and p18 have been localized within the cell by immunofluorescence methods and appear to be present throughout the kDNA. Electron-microscopic immunolocalization of p16 shows that p16 is present both within the kDNA disc and in the mitochondrial matrix at opposite edges of the kDNA disc. Our results suggest that nucleus-encoded H1-like proteins may be involved in the organization and segregation of kDNA networks in trypanosomatids.

The Crithidia fasciculata RNH1 gene encodes both nuclear and mitochondrial isoforms of RNase H.

The Crithidia fasciculata RNH1 gene encodes an RNase H, an enzyme that specifically degrades the RNA strand of RNA-DNA hybrids. The RNH1 gene is contained within an open reading frame (ORF) predicted to encode a protein of 53.7 kDa. Previous work has shown that RNH1 expresses two proteins: a 38 kDa protein and a 45 kDa protein which is enriched in kinetoplast extracts. Epitope tagging of the C-terminus of the RNH1 gene results in localization of the protein to both the kinetoplast and the nucleus. Translation of the ORF beginning at the second in-frame methionine codon predicts a protein of 38 kDa. Insertion of two tandem stop codons between the first ATG codon and the second in-frame ATG codon of the ORF results in expression of only the 38 kDa protein and the protein localizes specifically to the nucleus. Mutation of the second methionine codon to a valine codon prevents expression of the 38 kDa protein and results in exclusive production of the 45 kDa protein and localization of the protein only in the kinetoplast. These results suggest that the kinetoplast enzyme results from processing of the full-length 53.7 kDa protein. The nuclear enzyme appears to result from translation initiation at the second in-frame ATG codon. This is the first example in trypanosomatids of the production of nuclear and mitochondrial isoforms of a protein from a single gene and is the only eukaryotic gene in the RNase HI gene family shown to encode a mitochondrial RNase H.

Outcome of ABO-Incompatible Living Donor Renal Transplantations: A Single-Center Experience From Eastern India.

BACKGROUND: With the incessantly increasing number of patients on the waiting list for renal transplants, crossing the blood group barrier can substantially increase the donor pool. We started ABO-incompatible (ABOi) renal transplantation in 2013 with a relatively low-cost preconditioning protocol (additional cost, ∼$1200). This study reports the short-term outcome of ABOi renal transplantations performed at our institution. METHODS: A total of 45 adult ABOi kidney transplant recipients (KTRs) were included in the study. All of them underwent a pretransplantation conditioning program, which included plasmapheresis, low-dose intravenous immunoglobulin (IVIG), and low-dose rituximab. A pretransplantation isoagglutinin titer of ≤1:8 was considered acceptable for transplantation until December 2014, after which the threshold was increased to ≤1:32. RESULTS: Overall, 50% of KTRs were of blood group O. The maximum initial antibody titer was 1:2048. All the patients achieved immediate graft function post-transplantation. The mean serum creatinine level at 370 days (median duration of follow-up) was 1.21 mg%. One graft was lost due to severe antibody-mediated rejection (ABMR) with cortical necrosis. The graft survival rate was 97.78% and the patient survival rate was 97.78%. The overall result in terms of graft and patient survival, infections, and rejections were similar to ABO-compatible transplantations. CONCLUSIONS: ABOi renal transplantation is a cost-effective modality to increase the donor pool. Contrary to the belief that this modality is extremely expensive and requires elaborate infrastructure, we had a good short-term outcome with a relatively simple and low-cost preconditioning protocol.

Interference between M13 and oriM13 plasmids is mediated by a replication enhancer sequence near the viral strand origin.

The origin of replication for the viral strand of bacteriophage M13 DNA is contained within a 507 base-pair intergenic region of the phage chromosome. The viral strand origin is defined as the specific site at which the M13 gene II protein nicks the duplex replicative form of M13 DNA to initiate rolling-circle synthesis of progeny viral DNA. Using in vitro techniques we have constructed deletion mutations in M13 DNA at the unique AvaI site which is located 45 nucleotides away on the 3' side of the gene II protein nicking site. This deletion analysis has identified a sequence near the viral strand origin that is required for efficient replication of the M13 genome. We refer to this part of the intergenic region as a "replication enhancer" sequence. We have also studied the function of this sequence in chimeric pBR322-M13 plasmids and found that plasmids carrying both the viral strand origin and the replication enhancer sequence interfere with M13 phage replication. Based upon these findings we propose a model for the mechanism of action of the replication enhancer sequence involving binding of the M13 gene II protein.

Replication of the single-stranded DNA of the male-specific bacteriophage M13: circular forms of the replicative DNA.

Equilibrium centrifugation in caesium chloride, band sedimentation in alkaline solutions and electron microscopy have been used to study purified preparations of the M13 replicative DNA. The buoyant density of the M13 replicative DNA in a CsCl density gradient is 1.701, compared to a density of 1-710 for Escherichia coli DNA. When the replicative DNA is sedimented in alkaline solutions of CsCl (p = 1.35), two components are observed with uncorrected s-values of 48.6 +/- 0.2 and 15.2 +/- 0.5. Treatment of the replicative DNA with pancreatic DNase reduces the relative amount of the fast component in alkaline CsCl and similarly increases the relative amount of the slow component. Electron microscopic observation of the replicative DNA also shows two different forms of DNA, extended circles and condensed forms of DNA. The relative amount of condensed forms of DNA in a preparation of replicative DNA is equal to the relative amount of the fast component in alkaline velocity sedimentation. The average contour length of the extended circles is 2.19 +/- 0.07 micro.

Replication of the single-stranded DNA of the male-specific bacteriophage M13. Isolation of intracellular forms of phage-specific DNA.

The intracellular DNA has been isolated from Escherichia coli F+ cells infected with the small male-specific bacteriophage M13 and fractionated on a methylated albumin-kieselguhr column. Two infectious forms of phage-specific DNA were isolated and identified as a double-stranded replicative form and single-stranded DNA on the basis of their elution from the methylated albumin-kieselguhr column, infectivity to spheroplasts before and after heating, sedimentation at low ionic strength and buoyant density in CsCl. The replicative DNA was found to be resistant to thermal denaturation, suggesting that it may be a closed ring. It is estimated that there are at least 130 molecules of replicative DNA and 190 molecules of single-stranded DNA per infected bacterium.

Construction and characterization of new coliphage M13 cloning vectors.

New single-stranded DNA cloning vectors have been constructed by the insertion of additional DNA fragments into a HaeII restriction site in the bacteriophage M13 duplex replicative form (RF). These inserts into the M13 genome bring a single restriction sites useful for cloning, including PstI, XorII, EcoRI, SstI, XhoI, KpnI, and PvuII. Drug-resistance genes cloned into M13 include the beta-lactamase (bla) gene and the chloramphenicol acetyl transferase (cat) gene. These vectors provide a convenient means of easily obtaining the separated strands of a cloned duplex DNA fragment by cloning the fragment in each of the two possible orientations. Standard cloning techniques commonly applied to double-stranded DNAs can be utilized to insert foreign DNAs into the duplex RF DNAs of these vectors. Cells transformed by chimeric DNAs extrude filamentous phage particles carrying a circular single-stranded copy of the chimeric viral strand. Because M13-infected cells continue to grow and divide, cells can be transformed to yield either plaques or drug-resistant colonies. Specific inserts are readily detected by plaque hybridization techniques using an appropriate probe. Chimeric viral single strands from virus particles in the supernatant of small volumes of infected cultures can be rapidly and sensitively analyzed by agarose gel electrophoresis to determine the size of an insert.

One strand of ars189 from the maxicircle of Crithidia fasciculata transforms Saccharomyces cerevisiae more efficiently than its complementary strand as a single stranded DNA.

An autonomously replicating element (ars189) has been isolated from the maxicircle DNA of an insect trypanosomatid Crithidia fasciculata. This 189-bp fragment contains two copies of the yeast consensus ARS sequence of (A/T)TTTATPuTTT(T/A), has an A + T composition of 79.4%, and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands. The complementary strands of ars189 have been cloned into an M13 vector containing the URA3 gene of Saccharomyces cerevisiae. When these circular single-stranded (ss) DNAs were used to transform yeast spheroplasts, the M13 chimeric DNA carrying the strand of ars189 rich in adenine generated approximately four times more yeast Ura + transformants than the construct containing the thymine-rich strand. In contrast, both strands of yeast ARS1 cloned into an M13 vector transformed yeast at an equivalent level. The conversion of ARS-containing ss DNAs to duplex forms in vivo and their subsequent autonomous replication have been verified by Southern hybridization analysis of extracts from yeast transformants.

Conservation of a 29-base-pair sequence within maxicircle ARS fragments from six species of trypanosomes.

The maxicircles from Trypanosoma brucei, Herpetomonas samuelpessoai, Leptomonas seymouri, and Phytomonas davidi were examined for the presence of a 29-bp sequence termed CF29 that has been found in the ars 189 sequence from the Crithidia fasciculata maxicircle and in Lt-ars 189 from the maxicircle of Leishmania tarentolae. The CF29 sequence also contains a yeast consensus ARS of (T/A)TTTATPuTTT(T/A). All of the maxicircles examined contained specific fragments that hybridized to the CF29 probe. The non-replicating yeast plasmid vector YIp5 was used to clone these CF29-containing maxicircle fragments. High-frequency transformation was observed when these chimeric plasmids were used to transform Saccharomyces cerevisiae. Autonomous replication of these transforming plasmids was verified by Southern analysis of yeast-cell extracts using pBR322 as a hybridization probe. Therefore it appears that the CF29 sequence is widely conserved in kinetoplastid protozoa and is associated with ARS sequences in the maxicircles. Hybridization of the CF29 probe to a population of P. davidi minicircles was also observed. However, the YIp5 chimeric plasmid containing this CF29-hybridizing minicircle fragment failed to transform yeast.

Insertion of the Tn3 transposon into the genome of the single-stranded DNA phage M13.

The transposable genetic element Tn3, which carries an ampicillin (Ap) resistance determinant, has been translocated from a ColE1-Apr plasmid, RSF2124, to the genome of the filamentous single-stranded DNA phage M13. The site orientation of the inserted element has been determined for one such phage, M13::Tn3-15. The insertion is within the intergenic space separating genes 2 and 4 and containing both the viral strand and complementary strand origins. The lengths of both the filamentous phage and the duplex replicative form (RF) DNA are 1.7--1.8 times those of M13 phage and replicative form DNA. Both plaque formation and transduction of sensitive cells to ampicillin resistance by M13::Tn3-15 are sensitive to purified antibodies to the M13 major coat protein.

A 189-bp fragment of Crithidia fasciculata maxicircle DNA confers autonomous replication in Saccharomyces cerevisiae.

A 189-bp fragment capable of promoting high-frequency transformation in Saccharomyces cerevisiae has been isolated from the maxicircle of the insect trypanosomatid Crithidia fasciculata. Chimeric plasmids containing this autonomously replicating sequence (ars) are maintained as extrachromosomal elements in S. cerevisiae. The nucleotide sequence of the maxicircle fragment, termed ars189, has been determined, and its position has been mapped in the maxicircle. The ars189 fragment has an A + T content of 79.4% and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands. In one strand (the T strand) thymine accounts for 118 out of 189 nucleotides while adenine accounts for only 32 nucleotides. The ars189 DNA does not hybridize with minicircles, and its sequence appears to be unique in the C. fasciculata maxicircle genome. This sequence also shows extensive homology to a sequence within a 2.6-kb ars fragment of the Leishmania tarentolae maxicircle. In addition, ars189 contains two copies of a yeast consensus ars sequence (A/T)TTTATPuTTT(T/A).

High-level expression of M13 gene II protein from an inducible polycistronic messenger RNA.

Bacteriophage M13 gene II has been cloned in the plasmid expression vector pING1 and thereby placed under the control of the inducible araB promoter of Salmonella typhimurium. Upon induction with arabinose, gene II is transcribed as part of a polycistronic messenger RNA which initiates at the araB promoter. Subsequent translation of this message results in the coordinate, high-level expression of several proteins, including the gene II protein. Using this expression system, we have been able to overproduce gene II protein to a level of almost 15% of the total protein in Escherichia coli cells, thus providing an abundant source for its purification.