Swyer–James–MacLeod syndrome is characterised by unilateral hyperlucency on chest radiograph with small or normal-sized lung on the affected side and compensatory hyperinflation of opposite lung. Hyperinflation of the affected lung is a very rarely reported entity. An adult female patient, who presented with exertional breathlessness and diagnosed to have hypoplastic left pulmonary artery with hyperlucent, hyperinflated and herniated left lung is described.
Congenital muscular torticollis results from shortening or excessive contraction of the sternocleidomastoid (SCM) muscle. The reported incidence varies between 0.4 and 1.9%. Various theories have been proposed, but its true aetiology remains obscure. The deformity is characterized by a practically painless, contracted cordlike SCM muscle, which pulls the head toward the side affected, narrows and draws the shoulder upward, forcing the chin in the opposite direction. Torticollis of congenital origin is a deformity not commonly met with in the practice of maxillofacial surgery. The rarity, lack of, or inadequacy of the previous treatment, together with the advanced age and marked deformity appear to warrant an investigation and report of the outcome. A case of congenital muscular torticollis is presented who reported at the age of 18 years and has been successfully treated by unipolar SCM release.
The aim of our study was to evaluate the advantages and disadvantages of 3D plating system in the treatment of mandibular fractures.
Patients and Methods:
20 mandibular fractures in 18 patients at various anatomic locations and were treated by open reduction and internal fixation using 3D plates. All patients were followed at regular intervals of 4th, 8th and 12th weeks respectively. Patients were assessed post-operatively for lingual splay and occlusal stability. The incidence of neurosensory deficit, infection, masticatory difficulty, non-union, malunion was also assessed.
A significant reduction in lingual splay (72.2%) and occlusal stability (72.2%) was seen. The overall complication rate was (16.6%) which included two patients who developed post-operative paresthesia of lip, three patients had infection and two cases of masticatory difficulty which later subsided by higher antibiotics and 4 weeks of MMF. No evidence of non-union, malunion was noted.
A single 3D 2 mm miniplate with 2 mm × 8 mm screws is a reliable and an effective treatment modality for mandibular fracture.
3D plates; mandible fractures; miniplates; lingual splay
This study shows that antifungal curcumin (CUR), significantly depletes ergosterol levels in Candida albicans. CUR while displaying synergy with fluconazole (FLC) lowers ergosterol. However, CUR alone at its synergistic concentration (lower than MIC50), could not affect ergosterol contents. For deeper insight of CUR effects on lipids, we performed high throughput mass spectroscopy (MS) based lipid profiling of C. albicans cells. The lipidome analysis revealed that there were no major changes in PGLs composition following CUR treatment of Candida, however, significant differences in molecular species of PGLs were detected. Among major SPLs, CUR treatment resulted in the reduction of ceramide and accumulation of IPCs levels. The lipidome of CUR treated cells confirmed a dramatic drop in the ergosterol levels with a simultaneous accumulation of its biosynthetic precursors. This was further supported by the fact that the mutants defective in ergosterol biosynthesis (ERG2 and ERG11) and those lacking the transcription factor regulating ergosterol biosynthesis, UPC2, were highly susceptible to CUR. Our study first time shows that CUR, for its antifungal activity, targets and down regulates Δ5, 6 desaturase (ERG3) resulting in depletion of ergosterol. This results in parallel accumulation of ergosterol biosynthetic precursors, generation of ROS and cell death.
Candida albicans; Curcumin; Ergosterol; Δ5,6; desaturase Lipid homeostasis
Background & objectives:
ADAM33 is a member of a family of genes that encode membrane-anchored proteins with a disintegrin and a metalloprotease domain, primarily expressed in lung fibroblasts and bronchial smooth muscle cells. ADAM33 has been identified as a risk factor for asthma and is known as a gene associated with airway remodelling. The present study was conducted with the aims to investigate the expression of ADAM33 protein in patients of asthma and non-asthmatic controls, and to assess if the expression of ADAM33 protein relates with severity of asthma.
A total of 35 subjects, including 27 patients with asthma and eight non-asthmatic controls were included using Global Initiative for Asthma guidelines 2005. Bronchial biopsy tissues were collected and paraffin sections were made to store all study samples. Immunohistochemistry was performed using standardized protocol.
An increase in expression of ADAM33 protein was observed in the epithelium, smooth muscle and mesenchymal cells of asthma cases when compared to controls but there was no relationship with severity of asthma.
Interpretation & conclusions:
A higher expression of ADAM33 protein was seen in asthma patients compared to controls. Large prospective studies need to be done with adequate study design to confirm these preliminary finding.
ADAM33 protein expression; asthma; bronchial biopsy; immunohistochemistry; severity of asthma
Previous studies have reported that the dental follicular tissues associated with impacted lower third molars (ILTMs) may undergo cystic degeneration and/or neoplastic transformation. This is especially likely when the pericoronal space is >2.5 mm on intraoral radiographs and >3 mm on panoramic radiographs and to examine dental follicular tissue for pathological changes in patients with ILTMs and pericoronal radiolucencies of <2.5 mm.
Histopathological evaluation of follicular tissues associated with ILTMs.
Materials and Methods:
The morphology of the hematoxylin and eosin-stained follicular tissues of 146 such impactions were studied.
On microscopy, no cystic structures with fibrous walls were identified. 85 cases (58%) showed fibrous or myxomatous connective tissue and no epithelial elements. 61 cases (42%) showed epithelial elements in addition to fibrocollagenous tissue. Of these, 16 cases exhibited epithelium, of which 13 cases showed reduced enamel epithelium and three cases showed squamous metaplasia/non-keratinized stratified squamous epithelium.
All asymptomatic unerupted third molars with pericoronal radiolucency of <2.5 mm should be retained since they do not exhibit cyst formation microscopically.
Dental follicle; impacted lower third molar; pathology
Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix–hairpin–helix [(HhH)2] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)2 domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo.
To better understand alkylating agent-induced cytotoxicity and the base lesion DNA repair process in Saccharomyces cerevisiae, we replaced the RAD27FEN1 open reading frame (ORF) with the ORF of the bifunctional human repair enzyme DNA polymerase (Pol) β. The aim was to probe the effect of removal of the incised abasic site 5′-sugar phosphate group (i.e., 5′-deoxyribose phosphate or 5′-dRP) in protection against methyl methanesulfonate (MMS)-induced cytotoxicity. In S. cerevisiae, Rad27Fen1 was suggested to protect against MMS-induced cytotoxicity by excising multinucleotide flaps generated during repair. However, we proposed that the repair intermediate with a blocked 5′-end, i.e., 5′-dRP group, is the actual cytotoxic lesion. In providing a 5′-dRP group removal function mediated by dRP lyase activity of Pol β, the effects of the 5′-dRP group were separated from those of the multinucleotide flap itself. Human Pol β was expressed in S. cerevisiae, and this partially rescued the MMS hypersensitivity observed with rad27fen1-null cells. To explore this rescue effect, altered forms of Pol β with site-directed eliminations of either the 5′-dRP lyase or polymerase activity were expressed in rad27fen1-null cells. The 5′-dRP lyase, but not the polymerase activity, conferred the resistance to MMS. These results suggest that after MMS exposure, the 5′-dRP group in the repair intermediate is cytotoxic and that Rad27Fen1 protection against MMS in wild-type cells is due to elimination of the 5′-dRP group.
During mammalian base excision repair (BER) of lesion-containing DNA, it is proposed that toxic strand-break intermediates generated throughout the pathway are sequestered and passed from one step to the next until repair is complete. This stepwise process is termed substrate channeling. A working model evaluated here is that a complex of BER factors may facilitate the BER process. FLAG-tagged DNA polymerase (pol) β was expressed in mouse fibroblasts carrying a deletion in the endogenous pol β gene, and the cell extract was subjected to an ‘affinity-capture’ procedure using anti-FLAG antibody. The pol β affinity-capture fraction (ACF) was found to contain several BER factors including polymerase-1, X-ray cross-complementing factor1-DNA ligase III and enzymes involved in processing 3′-blocked ends of BER intermediates, e.g. polynucleotide kinase and tyrosyl-DNA phosphodiesterase 1. In contrast, DNA glycosylases, apurinic/aprymidinic endonuclease 1 and flap endonuclease 1 and several other factors involved in BER were not present. Some of the BER factors in the pol β ACF were in a multi-protein complex as observed by sucrose gradient centrifugation. The pol β ACF was capable of substrate channeling for steps in vitro BER and was proficient in in vitro repair of substrates mimicking a 3′-blocked topoisomerase I covalent intermediate or an oxidative stress-induced 3′-blocked intermediate.
Drug susceptible clinical isolates of Candida albicans frequently become highly tolerant to drugs during chemotherapy, with dreadful consequences to patient health. We used RNA sequencing (RNA-seq) to analyze the transcriptomes of a CDR (Candida Drug Resistance) strain and its isogenic drug sensitive counterpart.
RNA-seq unveiled differential expression of 228 genes including a) genes previously identified as involved in CDR, b) genes not previously associated to the CDR phenotype, and c) novel transcripts whose function as a gene is uncharacterized. In particular, we show for the first time that CDR acquisition is correlated with an overexpression of the transcription factor encoding gene CZF1. CZF1 null mutants were susceptible to many drugs, independently of known multidrug resistance mechanisms. We show that CZF1 acts as a repressor of β-glucan synthesis, thus negatively regulating cell wall integrity. Finally, our RNA-seq data allowed us to identify a new transcribed region, upstream of the TAC1 gene, which encodes the major CDR transcriptional regulator.
Our results open new perspectives of the role of Czf1 and of our understanding of the transcriptional and post-transcriptional mechanisms that lead to the acquisition of drug resistance in C. albicans, with potential for future improvements of therapeutic strategies.
Candida albicans; Multidrug resistance; Yeast; RNA-seq; CZF1
In this study, we show that a chemical dye, malachite green (MG), which is commonly used in the fish industry as an antifungal, antiparasitic, and antibacterial agent, could effectively kill Candida albicans and non-C. albicans species. We have demonstrated that Candida cells are susceptible to MG at a very low concentration (MIC that reduces growth by 50% [MIC50], 100 ng ml−1) and that the effect of MG is independent of known antifungal targets, such as ergosterol metabolism and major drug efflux pump proteins. Transcriptional profiling in response to MG treatment of C. albicans cells revealed that of a total of 207 responsive genes, 167 genes involved in oxidative stress, virulence, carbohydrate metabolism, heat shock, amino acid metabolism, etc., were upregulated, while 37 genes involved in iron acquisition, filamentous growth, mitochondrial respiration, etc., were downregulated. We confirmed experimentally that Candida cells exposed to MG resort to a fermentative mode of metabolism, perhaps due to defective respiration. In addition, we showed that MG triggers depletion of intracellular iron pools and enhances reactive oxygen species (ROS) levels. These effects could be reversed by the addition of iron or antioxidants, respectively. We provided evidence that the antifungal effect of MG is exerted through the transcription regulators UPC2 (regulating ergosterol biosynthesis and azole resistance) and STP2 (regulating amino acid permease genes). Taken together, our transcriptome, genetic, and biochemical results allowed us to decipher the multiple mechanisms by which MG exerts its anti-Candida effects, leading to a metabolic shift toward fermentation, increased generation of ROS, labile iron deprivation, and cell necrosis.
Prolonged usage of antifungal azoles which target enzymes involved in lipid biosynthesis invariably leads to the development of multi-drug resistance (MDR) in Candida albicans. We had earlier shown that membrane lipids and their fluidity are closely linked to the MDR phenomenon. In one of our recent studies involving comparative lipidomics between azole susceptible (AS) and azole resistant (AR) matched pair clinical isolates of C. albicans, we could not see consistent differences in the lipid profiles of AS and AR strains because they came from different patients and so in this study, we have used genetically related variant recovered from the same patient collected over a period of 2-years. During this time, the levels of fluconazole (FLC) resistance of the strain increased by over 200-fold. By comparing the lipid profiles of select isolates, we were able to observe gradual and statistically significant changes in several lipid classes, particularly in plasma membrane microdomain specific lipids such as mannosylinositolphosphorylceramides and ergosterol, and in a mitochondrial specific phosphoglyceride, phosphatidyl glycerol. Superimposed with these quantitative and qualitative changes in the lipid profiles, were simultaneous changes at the molecular lipid species levels which again coincided with the development of resistance to FLC. Reverse transcriptase-PCR of the key genes of the lipid metabolism validated lipidomic picture. Taken together, this study illustrates how the gradual corrective changes in Candida lipidome correspond to the development of FLC tolerance. Our study also shows a first instance of the mitochondrial membrane dysfunction and defective cell wall (CW) in clinical AR isolates of C. albicans, and provides evidence of a cross-talk between mitochondrial lipid homeostasis, CW integrity and azole tolerance.
Base excision repair (BER) can protect a cell after endogenous or exogenous genotoxic stress, and a deficiency in BER can render a cell hypersensitive to stress-induced apoptotic and necrotic cell death, mutagenesis, and chromosomal rearrangements. However, understanding of the mammalian BER system is not yet complete as it is extraordinarily complex and has many back-up processes that complement a deficiency in any one step. Due of this lack of information, we are unable to make accurate predictions on therapeutic approaches targeting BER. A deeper understanding of BER will eventually allow us to conduct more meaningful clinical interventions. In this review, we will cover historical and recent information on mammalian BER and DNA polymerase β and discuss approaches toward development and use of small molecule inhibitors to manipulate BER. With apologies to others, we will emphasize results obtained in our laboratory and those of our collaborators.
Base excision repair; DNA polymerase β; PARP-1; Small molecule inhibitors; PARP inhibitors; Structural biology; Mouse models; DNA repair deficiency
Overexpression of the CaCDR1-encoded multidrug efflux pump protein CaCdr1p (Candida drug resistance protein 1), belonging to the ATP binding cassette (ABC) superfamily of transporters, is one of the most prominent contributors of multidrug resistance (MDR) in Candida albicans. Thus, blocking or modulating the function of the drug efflux pumps represents an attractive approach in combating MDR. In the present study, we provide first evidence that the quorum-sensing molecule farnesol (FAR) is a specific modulator of efflux mediated by ABC multidrug transporters, such as CaCdr1p and CaCdr2p of C. albicans and ScPdr5p of Saccharomyces cerevisiae. Interestingly, FAR did not modulate the efflux mediated by the multidrug extrusion pump protein CaMdr1p, belonging to the major facilitator superfamily (MFS). Kinetic data revealed that FAR competitively inhibited rhodamine 6G efflux in CaCdr1p-overexpressing cells, with a simultaneous increase in an apparent Km without affecting the Vmax values and the ATPase activity. We also observed that when used in combination, FAR at a nontoxic concentration synergized with the drugs at their respective nonlethal concentrations, as was evident from their <0.5 fractional inhibitory concentration index (FICI) values and from the drop of 14- to 64-fold in the MIC80 values in the wild-type strain and in azole-resistant clinical isolates of C. albicans. Our biochemical experiments revealed that the synergistic interaction of FAR with the drugs led to reactive oxygen species accumulation, which triggered early apoptosis, and that both could be partly reversed by the addition of an antioxidant. Collectively, FAR modulates drug extrusion mediated exclusively by ABC proteins and is synergistic to fluconazole (FLC), ketoconazole (KTC), miconazole (MCZ), and amphotericin (AMB).
We recently demonstrated that CDR1 overexpression in azole-resistant isolates of Candida albicans is due to its enhanced transcriptional activation and increased mRNA stability. In this study, we provide the first evidence of transcriptional regulation of CDR1 by Ncb2, the β subunit of NC2, a heterodimeric regulator of transcription. Conditional NCB2 null mutants displayed decreased susceptibility toward azole and an enhanced transcription of CDR1. Interestingly, Ncb2 associated with the CDR1 promoter under both repression and activation; however, an increase in recruitment was observed under both transient and constitutive activation states. By chromatin immunoprecipitation (ChIP) assay, we showed the preferential recruitment of Ncb2 to the core TATA region under activation (azole-resistant isolate), while under repression (azole-susceptible isolate) it was present at the TATA upstream region. Further, ChIP analysis revealed that Ncb2 binding was not restricted to the CDR1 gene; instead, it was observed on the promoters of genes coregulated with CDR1 by the transcription activator Tac1. The tac1Δ null mutants, which fail to show the drug-induced transient activation of CDR1, also showed no increase in Ncb2 recruitment at the promoter. Taken together, our results show that Ncb2, in conjunction with Tac1, is involved in the transcriptional activation of CDR1, opening up new therapeutic possibilities to combat multidrug resistance (MDR) in C. albicans.
To study the role of urinary enzymes N-acetyl-β-glucosaminidase (NAG), alkaline phosphatase (AKP) and gamma glutamyl transferase (GGT) in the diagnosis and follow-up of patients with suspected pelviureteric junction obstruction (PUJO).
Materials and Methods:
A total of 70 patients, 29 managed conservatively (group A) and 41 managed by pyeloplasty (group B), were studied prospectively. A serial measurement of urinary enzymes NAG, AKP and GGT level was performed in both the groups. The mean levels of these urinary enzymes were compared between the two groups and among the patients of the same group at presentation as well as during follow-up.
There was a significant fall in the mean AKP level in patients managed conservatively at 8 months of follow-up. Similarly, in the operated group, there was a significant fall in the AKP levels at both 3 months and 8 months of follow-up. The mean level of GGT also showed a significant fall after 3 months of surgery but did not show further significant change at 8 months after surgery. The mean levels of NAG and GGT in the conservatively managed group were significantly low compared with that of patients requiring pyeloplasty at presentation as well as in the follow-up. The mean level of AKP was significantly low in the conservatively managed group when compared with the patients requiring surgery, but did not differ significantly in both the follow-ups after surgery.
The level of urinary enzymes NAG, AKP and GGT are significantly high in the patients with hydronephrosis (HDN) requiring pyeloplasty when compared with the patients managed conservatively. The level of AKP significantly falls after pyeloplasty in the patients of HDN due to PUJO. There is a negative correlation with the preoperative level of enzyme NAG with split renal function in the patients of HDN requiring pyeloplasty.
Alkaline phosphatase; gamma glutamyl transferase; N-acetyl-β-glucosaminidase; pelviureteric junction obstruction; urinary biomarkers; urinary enzymes
This study was designed to compare the pattern of obstructive sleep apnea (OSA) among obese and nonobese subjects regarding clinical and polysomnographic data obtained for a polysomnographic study.
A cross-sectional retrospective descriptive study was conducted by analyzing polysomnographic data in 112 consecutive patients underwent a sleep study at our sleep laboratory from January 2009 to July 2010. Out of them, 81 were diagnosed to have OSA (apnea-hypopnoea Index ≥5). These patients were classified in two groups with body mass index (BMI) < 27.5 kg/m2 as nonobese and BMI≥27.5 kg/m2 as obese. Clinical as well as polysomnographic data were evaluated and compared between the two groups. Patients were also evaluated for other risk factors such as smoking, alcoholism, and use of sedatives. Data were subjected to statistical analysis (χ2-test, P value <0.05 considered to be significant). The Fisher Exact test was applied wherever the expected frequency for a variable was ≤5.
Of 81 patients with OSA, 36 (44.4%) were nonobese with a mean BMI of 26.62 ± 2.29 kg/m2 and 45 (55.6%) were obese with a mean BMI of 35.14 ± 3.74 kg/m2. Mean AHI per hour was significantly more in the obese than in the nonobese group (50.09 ± 29.49 vs. 24.36 ± 12.17, P<0.001). The use of one or more sedatives was more in nonobese as compared to obese (58.3% vs. 24.4%, P=0.002). The obese group had significantly higher desaturation and arousal index (P<0.001). The minimal oxygen saturation was lower in the obese than the nonobese group (68.5 ± 13.00 vs. 80.3 ± 7.40, P<0.001) and was well below 90% in both groups. Overall, the OSA in nonobese patients was mild-to-moderate as compared to that of the obese and no significant differences were observed between them as regard to age, gender, mean neck circumference, excessive daytime sleepiness, adenoid or tonsillar enlargement, smoking, and remaining polysomnographic parameters.
Obstructive sleep apnea can occur in nonobese persons though with less severity as compared to obese leading to a concept that OSA is not restricted to obese persons only and there is a high demand of its awareness regarding evaluation, diagnosis, and management in such individuals.
Body mass index; obesity; obstructive sleep apnea
The correction of skeletal Class III malocclusion with severe mandibular prognathism in an adult individual requires surgical and Othodontic combination therapy. The inter disciplinary approach is the treatment of choice in most of the skeletal malocclusions. A case report of an adult individual with Class III malocclusion, having mandibular excess in sagittal and vertical plane and treated with orthodontics,, bilateral sagittal split osteotomy and Le – Forte I osteotomy for the correction of skeletal, dental and soft tissue discrepancies is herewith presented. The surgical–orthodontic combination therapy has resulted in near–normal skeletal, dental and soft tissue relationship, with marked improvement in the facial esthetics in turn, has helped the patient to improve the self-confidence level.
Orthognathic Surgery; Bilateral sagittal split osteotomy; surgical orthodontics
The action of multidrug efflux pumps in MDR (multidrug resistance) acquisition has been proposed to partially depend on the transport of physiological substrates which may indirectly affect drug partition and transport across cell membranes. In the present study, the PDR18 gene [ORF (open reading frame) YNR070w], encoding a putative PDR (pleiotropic drug resistance) transporter of the ATP-binding cassette superfamily, was found to mediate plasma membrane sterol incorporation in yeast. The physiological role of Pdr18 is demonstrated to affect plasma membrane potential and is proposed to underlie its action as a MDR determinant, conferring resistance to the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid). The action of Pdr18 in yeast tolerance to 2,4-D, which was found to contribute to reduce [14C]2,4-D intracellular accumulation, may be indirect, given the observation that 2,4-D exposure deeply affects the sterol plasma membrane composition, this effect being much stronger in a Δpdr18 background. PDR18 activation under 2,4-D stress is regulated by the transcription factors Nrg1, controlling carbon source availability and the stress response, and, less significantly, Yap1, involved in oxidative stress and MDR, and Pdr3, a key regulator of the yeast PDR network, consistent with a broad role in stress defence. Taken together, the results of the present study suggest that Pdr18 plays a role in plasma membrane sterol incorporation, this physiological trait contributing to an MDR phenotype.
ergosterol homoeostasis; herbicide resistance; multidrug resistance; pleiotropic drug resistance; Saccharomyces cerevisiae; ABC, ATP-binding cassette; CM, cell membrane; 2,4-D, 2,4-dichlorophenoxyacetic acid; 2,4-DCP, 2,4-dichlorophenol; DiOC6(3), 3-3′-dihexyloxacarbocianine iodide; ER, endoplasmic reticulum; MCPA, 2-methyl-4-chlorophenoxyacetic acid; MDR, multidrug resistance; MFS, major facilitator superfamily; ORF, open reading frame; PDR, pleiotropic drug resistance; RT, reverse transcription
We explored DNA metabolic events potentially relevant to somatic hypermutation (SHM) of immunoglobulin genes using a yeast model system. Double-strand break (DSB) formation has been discussed as a possible component of the SHM process during immunoglobulin gene maturation. Yet, possible mechanisms linking DSB formation with mutagenesis have not been well understood. In the present study, a linkage between mutagenesis in a reporter gene and a double-strand break at a distal site was examined as a function of activation-induced deaminase (AID) expression. Induction of the DSB was found to be associated with mutagenesis in a genomic marker gene located 7kb upstream of the break site: mutagenesis was strongest with the combination of AID expression and DSB induction. The mutation spectrum of this DSB and AID-mediated mutagenesis was characteristic of replicative bypass of uracil in one strand and was dependent on expression of DNA polymerase delta (Pol δ). These results in a yeast model system illustrate that the combination of DSB induction and AID expression could be associated with mutagenesis observed in SHM. Implications of these findings for SHM of immunoglobulin genes in human B cells are discussed.
Activation-induced deaminase; AID; base excision repair; somatic hypermutation; double-strand breaks; mutagenesis
Background & objectives:
The mechanisms that protect female upper genital tract from ascending infection by microbes present in vagina are only partially understood. It is expected that epithelial cells in mucosal surfaces and their secretions directly interfere with microbial colonization and invasion. This study was aimed to demonstrate the expression of 2 kDa antimicrobial peptide which was identified and purified from female genital tract tissues using chromatographic techniques.
Low molecular weight proteins were isolated from human female reproductive tract tissues obtained from premenopausal women. Antimicrobial activity of these LMW proteins was assessed against different reproductive tract pathogens viz., Neisseria gonorrhoeae, Group B streptococcus, Gardnerella vaginalis, Escherechia coli and Candida albicans. The expression of these peptides were also documented in reproductive tract tissues with the help of hyperimmune sera raised against the rabbits. The purified peptide was characterized by N-terminal sequencing.
Immunohistochemical and immunofluorescence studies demonstrated that 2 kDa peptide was expressed in the stratified squamous epithelial cells of the ectocervix while it was absent in columnar epithelial cells of upper genital tract. Upregulation of the expression of this peptide was observed in patients of chronic non-specific cervicitis and acute on chronic cervicitis. This purified antimicrobial peptide also showed broad spectrum antimicrobial activity against different reproductive tract pathogens.
Interpretation & conclusions:
Considering the emerging bacterial resistance against conventional antibiotics, isolation and understanding of the expression of antimicrobial peptides from female reproductive tissue extracts may provide some leads towards the development of strategies for the treatment of reproductive tract infections.
Antimicrobials; antimicrobial peptides; female reproductive tract; innate immunity; sexually transmitted infection
The base excision DNA repair (BER) pathway known to occur in Caenorhabditis elegans has not been well characterized. Even less is known about the DNA polymerase (pol) requirement for the gap-filling step during BER. We now report on characterization of in vitro uracil-DNA initiated BER in C. elegans. The results revealed single-nucleotide (SN) gap-filling DNA polymerase activity and complete BER. The gap-filling polymerase activity was not due to a DNA polymerase β (pol β) homolog, or to another X-family polymerase, since computer-based sequence analyses of the C. elegans genome failed to show a match for a pol β-like gene or other X-family polymerases. Activity gel analysis confirmed the absence of pol β in the C. elegans extract. BER gap-filling polymerase activity was partially inhibited by both dideoxynucleotide and aphidicolin. The results are consistent with a combination of both replicative polymerase(s) and lesion bypass/BER polymerase pol θ contributing to the BER gap-filling synthesis. Involvement of pol θ was confirmed in experiments with extract from pol θ null animals. The presence of the SN BER in C. elegans is supported by these results, despite the absence of a pol β-like enzyme or other X-family polymerase.
Nucleotide Binding Domains (NBDs) of multidrug transporter of Candida albicans, CaCdr1p possess unique divergent amino acids in their conserved motifs. For example, NBD1 (N-terminal-NBD) possesses conserved Signature motifs while the same motif is divergent in NBD2 (C-terminal-NBD). In this study, we have evaluated the contribution of these conserved and divergent Signature motifs of CaCdr1p in ATP catalysis and drug transport. By employing site directed mutagenesis, we made three categories of mutant variants. These included mutants where all the Signature motif residues were either replaced with alanines or mutants with exchanged equipositional residues to mimic the conservancy and degeneracy in opposite domain. In addition, a set of mutants where Signature motifs were swapped to have variants with either both the conserved or degenerated entire Signature motif. We observed that conserved and equipositional residues of NBD1 and NBD2, and swapped Signature motif mutants showed high susceptibility to all the tested drugs with simultaneous abrogatation in ATPase and R6G efflux activities. However, some of the mutants displayed selective increase in susceptibility to the drugs. Notably, none of the mutant variants and WT-CaCdr1p showed any difference in drug and nucleotide binding. Our mutational analyses show that not only certain conserved residues of NBD1 Signature sequence (S304, G306 and E307) are important in ATP hydrolysis and R6G efflux but a few divergent residues (N1002 and E1004) of NBD2 Signature motif have also evolved to be functionally relevant and are not interchangeable. Taken together, our data suggest that the Signature motifs of CaCdr1p whether it is divergent or conserved, are non-exchangeable and functionally critical for ATP hydrolysis.
ABC transporter; Nucleotide Binding Domain; Signature motif; Drug resistance; Drug transport; ATPase activity
Verbal autopsy methods are critically important for evaluating the leading causes of death in populations without adequate vital registration systems. With a myriad of analytical and data collection approaches, it is essential to create a high quality validation dataset from different populations to evaluate comparative method performance and make recommendations for future verbal autopsy implementation. This study was undertaken to compile a set of strictly defined gold standard deaths for which verbal autopsies were collected to validate the accuracy of different methods of verbal autopsy cause of death assignment.
Data collection was implemented in six sites in four countries: Andhra Pradesh, India; Bohol, Philippines; Dar es Salaam, Tanzania; Mexico City, Mexico; Pemba Island, Tanzania; and Uttar Pradesh, India. The Population Health Metrics Research Consortium (PHMRC) developed stringent diagnostic criteria including laboratory, pathology, and medical imaging findings to identify gold standard deaths in health facilities as well as an enhanced verbal autopsy instrument based on World Health Organization (WHO) standards. A cause list was constructed based on the WHO Global Burden of Disease estimates of the leading causes of death, potential to identify unique signs and symptoms, and the likely existence of sufficient medical technology to ascertain gold standard cases. Blinded verbal autopsies were collected on all gold standard deaths.
Over 12,000 verbal autopsies on deaths with gold standard diagnoses were collected (7,836 adults, 2,075 children, 1,629 neonates, and 1,002 stillbirths). Difficulties in finding sufficient cases to meet gold standard criteria as well as problems with misclassification for certain causes meant that the target list of causes for analysis was reduced to 34 for adults, 21 for children, and 10 for neonates, excluding stillbirths. To ensure strict independence for the validation of methods and assessment of comparative performance, 500 test-train datasets were created from the universe of cases, covering a range of cause-specific compositions.
This unique, robust validation dataset will allow scholars to evaluate the performance of different verbal autopsy analytic methods as well as instrument design. This dataset can be used to inform the implementation of verbal autopsies to more reliably ascertain cause of death in national health information systems.
Verbal autopsy; VA; validation; Philippines; Tanzania; India; Mexico; gold standard; cause of death