Evaluation of Dual Dye Technique for Sentinel Lymph Node Biopsy in Breast Cancer: Two-Arm Open-Label Parallel Design Non-Inferiority Randomized Controlled Trial.

INTRODUCTION: Radioisotope and blue dye are standard agents for performing sentinel lymph node (SLN) biopsy in breast cancer. The paucity of nuclear medicine facility poses logistic challenge. This study evaluated performance of radioisotope & methylene blue (MB) with indocyanine green (ICG) and MB for SLNB. PATIENTS AND METHODS: This randomized controlled trial was conducted from December 2019 to July 2022 comparing SLN identification proportions of radioisotope-blue dye [Group A] with dual dye (MB + ICG; Group B]. Secondary objective included time required and cost effectiveness of performing SLNB. Sample size of 70 (35 in each arm) was calculated. Upfront operable node negative early breast cancer was included in the study. Clinico-demographic data, number & type of SLN, time taken were noted. Cost analysis was done including the equipment, manpower & consumables. Chi-square/Fisher exact test was used to compare proportion between two groups. p value of less than 0.05 was considered to represent statistical significance. RESULTS: Seventy patients randomized to either group were similar in clinico-demographic and tumor characteristics. SLN identification rate (IR) was 91.43% in group A and 100% in group B. Overall IR of MB, radioisotope and ICG were 91.43%, 91.43% and 100%, respectively. Mean number of SLNs identified were 3 in group A and 4 in group B. Median time required for SLNB was 12 min and 14 min in either group, respectively. Cost of performing SLNB was higher in Group B. CONCLUSION: SLNB using dual dye is non-inferior to radioisotope-blue dye in upfront operable early breast cancer. Trial registration number Clinical Trial registry India CTRI/2020/02/023503.

Radiology of anal and lower rectal cancers.

Lower rectal and anal cancers are distinct from neoplasms involving rest of the rectum. These are relatively difficult to manage owing to important relationships with the sphincter muscles. Involvement of the latter portends a poorer prognosis and increased chance of recurrence. Lymphatic drainage of these tumours is into the systemic circulation and the exact set of lymph nodes involved depends on the precise location of the tumour. The role of imaging includes assessment of local invasion, infiltration of adjacent pelvic organs, assessment of locoregional lymphatic spread and metastasis, post-chemoradiation restaging as well as post-treatment surveillance.

Microbial profile of the vitreous aspirates in culture proven exogenous endophthalmitis: A 10-year retrospective study.

PURPOSE: To describe the microbiological profile and clinical outcome in the eyes with culture-proven exogenous endophthalmitis. METHODS: A retrospective analysis of 495 eyes diagnosed as exogenous endophthalmitis was performed over a period of 10 years. In all, aseptically collected aqueous and vitreous aspirates were cultured for bacteria and fungus using standard microbiological techniques. Gram-stain and KOH preparation of the specimens were also performed. The antibiotic susceptibility testing for bacterial isolates was performed by Kirby-Bauer disk diffusion method. The treatment was modified according to the antibiotic sensitivity profile. The final clinical ocular condition was divided into improved, stable or deteriorated. RESULTS: Of 148 culture-proven endophthalmitis eyes, 137 (92.57%) were referred from elsewhere, and 11 (7.43%) belonged to our institute. Aetiologically, 76 (51.35%) eyes were post-cataract surgery, 61 (41.22%) were post-traumatic, 5 (3.38%) eyes post-intravitreal anti-vascular endothelial growth factor injection, 5 associated with corneal diseases and 1 bleb-related endophthalmitis. In 31 (20.95%) eyes, primary intravitreal antibiotics were given outside. The cultures revealed monomicrobial growth in 92.57% (n = 137) and polymicrobial growth in 7.43% (n = 11). Among the bacteria (n = 121, 81.76%), Pseudomonas species dominated overall (n = 32, 27.11%) and post-operative (n = 26, 38.23%) endophthalmitis group. Staphylococcus epidermidis (n = 14, 28%) was prominent in post-traumatic endophthalmitis group. Ninety-two percent (n = 108 isolates) of bacteria were sensitive to vancomycin. In 78 (52.7%) eyes, the clinical ocular condition improved or remained stable while deteriorated in 51 (34.46%). CONCLUSION: A bacterial predominance was observed among causative organisms of exogenous endophthalmitis with Pseudomonas species being the most common. The appropriate surgical intervention improved or stabilised the visual acuity in nearly 50% eyes.

Prevalence of the plasmid-mediated quinolone resistance determinants among clinical isolates of Shigella sp. in Andaman & Nicobar Islands, India.

AIMS: This study was carried out to find the prevalence of various plasmid-mediated quinolone-resistant (PMQR) determinants among the quinolone-resistant clinical isolates of Shigella sp. from paediatric patients in Andaman & Nicobar Islands. METHODS AND RESULTS: A total of 106 quinolone-resistant Shigella isolates obtained from paediatric patients during hospital-based surveillance from January 2003 to June 2010 were screened for the presence of various PMQR determinants. Of 106 isolates, 8 (7.5%) showed the presence of aac (6')-Ib-cr and 3 (2.8%) harboured the qnrB genes with 2 (1.9%) of these isolates showing the presence of both. All the 9 isolates had uniform mutations in gyrA (S83L) and in parC (S80I). CONCLUSIONS: The prevalence of fluoroquinolone-acetylating aminoglycoside acetyltransferase {aac (6')-Ib-cr} gene is higher than qnrB gene among the clinical Shigella isolates. These PMQR determinants were detected in the Shigella isolates obtained from 2008-2010, indicating that it happens in a stepwise manner following the multiple mutations in quinolone resistance-determining regions increase or extend resistance to quinolones or fluoroquinolones. SIGNIFICANCE AND IMPACT OF STUDY: The prevalence of these genes are of grave concern as it may be horizontally transferred to other human pathogenic bacteria and can lead to therapeutic failure as a consequence of antimicrobial resistance, not only for the islands but also for the entire south-east region. The results obtained should encourage further studies on the implications of the presence, distribution, association and variation of these determinants in our quest for understanding PMQR.

Experimental trial of transvaginal cholecystectomy: an ex vivo analysis of the learning process for a novel single-port technique.

BACKGROUND: Interest in natural orifice transluminal endoscopic surgery (NOTES) has expanded, and the first experiences with patients using different techniques have been reported. However, no work has addressed the learning process or the limitations of the procedures. The relation between inexperience and complications became a major concern after the introduction of laparoscopic surgery. This study investigates the learning process for a new technique using specially designed instruments in an ex vivo model before clinical application. METHODS: Specially designed instruments and a single-port technique using the Tuebingen Trainer were used to evaluate instrument and surgeon performance (learning curve) in terms of time and errors. A total of 90 procedures performed by three surgeons were evaluated. Group and individual learning curves were plotted. RESULTS: All the surgeons showed a reduction in both mean cholecystectomy time (subject A: 27.2 vs 16.6 min; subject B: 21.4 vs 19.22 min; subject C: 21 vs 19.7 min) and mean errors (subject A: 2.8 vs 1.6; subject B: 3.5 vs 2.6; subject C: 3.5 vs 2). A plateau was reached after approximately 15 procedures. Group learning curve analysis showed a significant reduction in time between the first group (mean, 24.97 +/- 5.8 min) and last group (mean, 19.30 +/- 3.09 min; F[1,28] = 11.83; p = 0.001) for 15 procedures, as well as reduced technical errors in the fifth group, from 3.7 +/- 1.65 to 1.6 +/- 1.04 (F[1,28] = 8.90; p < 0.01), demonstrating a learning effect. The number of optic and access port position changes were recorded, setting a standard for normal instrument performance. CONCLUSION: This study shows that the tasks of cholecystectomy can be learned safely in a reasonable number of simulations with the new instruments. Although this is a new technique, prior laparoscopic surgery experience is helpful. The technique offers an advantage over those using flexible endoscopes.

Total extra-peritoneal repair of groin hernia: prospective evaluation at a tertiary care center.

BACKGROUND: The laparoscopic repair of groin hernia is increasingly being used. However, the relative merits and demerits of laparoscopic repair are debatable. The present study was undertaken to evaluate the total extra-peritoneal (TEP) repair of groin hernia. METHODS: This prospective study was undertaken at a single surgical unit between January 2004 and June 2006. Consecutive patients with elective groin hernias were offered laparoscopic TEP repair. Indigenous balloon or telescopic dissection was used to create extra-peritoneal space. Polypropylene mesh was used in all of the patients and mesh fixation was performed with tackers. RESULTS: A total of 185 patients with age range 18-92 years were included; 180 were males. TEP repair was attempted in 298 groin hernias in 185 patients with a success rate of 89.5%. Indigenous balloon or telescopic dissection was used to create extra-peritoneal space. Thirty-one (31, 10.5%) TEP repairs were converted to transabdominal pre-peritoneal or open repair. Two patients developed recurrence during follow-up. CONCLUSION: TEP is an excellent technique for laparoscopic groin hernia repair, with acceptable rates of complication.

Genomic organization and chromosomal localization of the Asna1 gene, a mouse homologue of a bacterial arsenic-translocating ATPase gene.

The plasmid encoded ArsA ATPase in Escherichia coli is the catalytic component of an oxyanion pump that is responsible for resistance to arsenicals and antimonials. Arsenite or antimonite allosterically activates the ArsA ATPase activity. In this paper, we report the cloning and characterization of the mouse homologue (Asna1) of the bacterial arsA gene. The Asna1 gene encodes an open reading frame of 348 amino acids and exhibits 27% identity to the bacterial ArsA protein and 99% similarity to its human counterpart (hASNA-1). The Asna1 mRNA is a approximately 1.3 kb transcript and is present at high levels in kidney and testis, moderate levels in brain, liver, lung and skin, and low levels in heart, small intestine, spleen, stomach, and thymus. A negligible amount of Asna1 transcript is detected in skeletal muscle. We have also characterized the genomic structure of the Asna1 gene. The gene spans over 7 kb and consists of seven exons and six introns. All splice sites conform to the GT-AG rule, except for the splice donor site of intron 4 that is GC instead of GT. Fluorescence in situ hybridization indicates that the Asna1 gene is localized in the C3-D1 region of mouse chromosome 8.

Structure-function analysis of the ArsA ATPase: contribution of histidine residues.

The ArsA ATPase is the catalytic subunit of the ArsAB oxyanion pump in Escherichia coli that is responsible for extruding arsenite or antimonite from inside the cell, thereby conferring resistance. Either antimonite or arsenite stimulates ArsA ATPase activity. In this study, the role of histidine residues in ArsA activity was investigated. Treatment of ArsA with diethyl pyrocarbonate (DEPC) resulted in complete loss of catalytic activity. The inactivation could be reversed upon subsequent incubation with hydroxylamine, suggesting specific modification of histidine residues. ATP and oxyanions afforded significant protection against DEPC inactivation, indicating that the histidines are located at the active site. ArsA has 13 histidine residues located at position 138, 148, 219, 327, 359, 368, 388, 397, 453, 465, 477, 520, and 558. Each histidine was individually altered to alanine by site-directed mutagenesis. Cells expressing the altered ArsA proteins were resistant to both arsenite and antimonite. The results indicate that no single histidine residue plays a direct role in catalysis, and the inhibition by DEPC may be caused by steric hindrance from the carbethoxy group.

Structure-function relationships in an anion-translocating ATPase.

The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli. This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells. The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit ArsB. The complex is similar in many ways to ATP-binding cassette ('ABC') transporters, which typically have two groups of six transmembrane-spanning helical segments and two nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 transmembrane-spanning segments. ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter. The 63 kDa ArsA protein is a substrate-activated ATPase. It has two homologous halves, A1 and A2, which are clearly the result of an ancestral gene duplication and fusion. Each half has a consensus NBD. The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses. Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA. The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme. Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step. Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.

Role of conserved histidine residues in metalloactivation of the ArsA ATPase.

The ArsA ATPase is the catalytic subunit of a pump that is responsible for resistance to arsenicals and antimonials in Escherichia coli. Arsenite or antimonite allosterically activates the ArsA ATPase activity. ArsA homologues from eubacteria, archaea and eukarya have a signature sequence (DTAPTGHT) that includes a conserved histidine. The ArsA ATPase has two such conserved motifs, one in the NH2-terminal (Al) half and the other in the COOH-terminal (A2) half of the protein. These sequences have been proposed to be signal transduction domains that transmit the information of metal occupancy at the allosteric to the catalytic site to activate ATP hydrolysis. The role of the conserved residues His148 and His453, which reside in the A1 and A2 signal transduction domains respectively, was investigated by mutagenesis to create H148A, H453A or H148A/H453A ArsAs. Each altered protein exhibited a decrease in the Vmax of metalloid-activated ATP hydrolysis, in the order wild type ArsA>H148A>H453A>H148A/H453A. These results suggest that the histidine residues play a role in transmission of the signal between the catalytic and allosteric sites.

Mechanism of the ArsA ATPase.

The ArsAB ATPase confers metalloid resistance in Escherichia coli by pumping toxic anions out of the cells. This transport ATPase shares structural and perhaps mechanism features with ABC transporters. The ArsAB pump is composed of a membrane subunit that has two groups of six transmembrane segments, and the catalytic subunit, the ArsA ATPase. As is the case with many ABC transporters, ArsA has an internal repeat, each with an ATP binding domain, and is allosterically activated by substrates of the pump. The mechanism of allosteric activation of the ArsA ATPase has been elucidated at the molecular level. Binding of the activator produces a conformational change that forms a tight interface of the nucleotide binding domains. In the rate-limiting step in the overall reaction, the enzyme undergoes a slow conformational change. The allosteric activator accelerates catalysis by increasing the velocity of this rate-limiting step. We postulate that similar conformational changes may be rate-limiting in the mechanism of ABC transporters.

Capsulorhexis in intumescent cataract.

This technique of anterior capsulorhexis is for use in eyes with high intralenticular pressure and absence of red reflex as encountered in intumescent cataract. The initial steps of fashioning the anterior capsular flap and lens decompression are done under the microscope's high magnification and noncoaxial oblique illumination. Intralenticular pressure is controlled by filling the anterior chamber with a viscoelastic agent. Once the lens is decompressed, the capsulorhexis is completed with a capsule forceps and an endoilluminator is used as an oblique source of illumination. This technique allows controlled capsulorhexis in eyes with intumescent cataract.

UDP-galactose 4-epimerase from Kluyveromyces fragilis: reconstitution of holoenzyme structure after dissociation with parachloromercuribenzoate.

UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis (Kluyveromyces marxianus var. marxianus) is a homodimer of molecular mass 75 kDa/subunit and has one mol NAD firmly bound/dimer. The pathway for the assembly of the holoenzyme structure has been studied after dissociating the native epimerase with p-chloromercuribenzoate into inactive mercurated monomers. The process of dissociation was not associated with unfolding of the molecules. Reconstitution of the functional holoenzyme was done by reduction with dithiothreitol and addition of extra NAD. The reaction was thus followed to monitor maturation of the enzyme from the folded monomeric state. The reconstituted enzyme was similar to the native enzyme in terms of a number of physiochemical properties such as secondary, tertiary and quarternary structures, Km for the substrate UDP-galactose, reductive inhibition, interaction with the fluorophore 1-anilino 8-naphthalene sulphonic acid (ANS), etc. Reconstitution under low ionic strength buffer (I = 0.011) shows that the presence of NAD is essential for the formation of a dimeric structure. However, dimeric apoenzyme could also be stabilized under high ionic strength buffer (I = 0.1). Reactivation was strongly dependent on pH, being most effective at pH 8.1. Kinetic evidence suggested that, at low ionic strength, assembly of NAD over dimeric apoenzyme is the rate-limiting step in expressing catalytic activity. This process has a low energy of activation of 27.2 kJ/mol.

Spatial proximity of Cys113, Cys172, and Cys422 in the metalloactivation domain of the ArsA ATPase.

ArsA ATPase activity is allosterically activated by salts of the semimetal arsenic or antimony. Activation is associated with the presence of three cysteine residues in ArsA: Cys113, Cys172, and Cys422. To determine the distance between cysteine residues, wild type ArsA and ArsA proteins with cysteine to serine substitutions were treated with the bifunctional alkylating agent dibromobimane, which reacts with thiol pairs within 3-6 A of each other to form a fluorescent adduct. ArsA proteins in which single cysteine residues were altered by site-directed mutagenesis still formed fluorescent adducts. Proteins in which two of the three cysteine residues were substituted did not form fluorescent adducts. These results demonstrate that Cys113, Cys172, and Cys422 are in close proximity of each other. We propose a model in which As(III) or Sb(III) interacts with these three cysteines in a trigonal pyramidal geometry, forming a novel soft metal-thiol cage.

Role of cysteinyl residues in metalloactivation of the oxyanion-translocating ArsA ATPase.

The ArsA protein, the catalytic subunit of the oxyanion-translocating ATPase responsible for resistance to arsenicals and antimonials in Escherichia coli, is activated by arsenite or antimonite. Activation is associated with dimerization of the ArsA protein. Enzymatic activity was rapidly but reversibly inhibited by the sulfhydryl reagent methyl methanethiosulfonate, suggesting that at least one cysteinyl residue is required for catalytic activity. Each of the four cysteinyl residues in the ArsA protein, Cys26, Cys113, Cys172, and Cys422, were individually changed to seryl residues. The C26S protein had normal properties. Cells expressing the other three mutations lost resistance to arsenite and antimonite. The C113S, C172S, and C422S enzymes each had relatively normal Km values for ATP but reduced affinity for antimonite and arsenite. The Vmax of the activated enzymes ranged from very low for the C113S and C422S enzymes to near normal for the C172S enzyme. These results suggest a mechanism of activation by formation of a tricoordinate complex between Sb(III) or As(III) and the cysteine thiolates 113, 172, and 422.