Accuracy of the International Academy of Cytology Yokohama system of breast cytology reporting for fine needle aspiration biopsy of the breast in a dedicated breast care setting.

BACKGROUND: The International Academy of Cytology (IAC) Yokohama system is a recently proposed system for reporting breast cytology by fine needle aspiration biopsies (FNAB). Multiple studies are needed to confirm the risk of malignancy (ROM) of the various reporting categories of this system. The present article studies the accuracy of the IAC Yokohama system in our center. METHODS: Over a period of 1 year (September 2018-August 2019), all cases of breast masses assessed by FNAB and histological correlation were studied retrospectively. Sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) and overall accuracy of the IAC Yokohama system for diagnosing malignancy were assessed. The rates of malignancy (ROM) of each diagnostic category were also estimated. RESULTS: Three hundred and twenty-one FNABs had cyto-histological correlation. The percent sensitivity (with 95% Confidence Intervals) when the atypical, suspicious of malignancy and the malignant categories were regarded as positive for malignancy were 98.2% [95.5%, 99.5%], 96.0% [92.5%, 98.2%], and 86.7% [81.5%, 90.8%] respectively. The percent specificity (with 95% Confidence intervals) for the same categories in the same order were 59.5% [47.4%, 70.7%], 91.9% [83.2%, 97.0%], and 100% [95.1%, 100%] respectively. The area under curve (AUC) for diagnosing malignancy was 0.981[0.963, 0.993]. The ROM for the benign, atypical, suspicious of malignancy and malignant category were 8.3% [2.3%, 20.0%], 17.2% [5.8%, 35.8%], 77.8% [57.7%, 91.4%], and 100% [98.1%, 100%] respectively. CONCLUSION: The IAC Yokohama system is suitable for accurately reporting breast lesions on FNAB.

Cytological diagnosis of osteoblastoma of the talus: Case report with unusual presentation and diagnostic pitfalls.

Osteoblastoma is a rare bone tumor mostly affecting the young adults and commonly involving the spinal cord and long bones. Talus is the uncommon site of presentation, and if involved, then the neck is more commonly involved than the body of talus. The cytological diagnosis of osteoblastoma is limited, and to the best of our knowledge, its fine-needle aspiration (FNA) in the talus has still not been reported in the literature. The present case of osteoblastoma is, therefore, being reported due to the unusual presentation in elderly male in the body of talus and showing extensive involvement on X-ray. The case was initially diagnosed on FNA cytology excluding the possibility of giant-cell tumor and osteosarcoma. The case also highlights the importance of vigilant observation of subtle cytological features of this rare tumor which may be helpful in avoiding diagnostic pitfalls, especially at an uncommon site and with unusual presentation. An early precise diagnosis by cytology may be followed by appropriate treatment and thus avoiding any further complications.

Primary sinonasal lymphoma in immunocompetent patients: A 10 years retrospective clinicopathological study.

INTRODUCTION: Sinonasal tumors occur in the nasal cavity or paranasal sinuses (PNS). These tumors are rare and lymphomas are even rarer. Lymphoma of the nose and PNS may mimic benign processes and may manifest either in an isolated fashion or in conjunction with systemic diseases. B-cell lymphomas, a more favorable diagnosis, account for the majority of cases, whereas T-cell and extranodal natural killer lymphoma are associated with rapid disease progression and death. MATERIALS AND METHODS: All patients with sinonasal lymphomas who were nonreactive for HIV and were operated and treated in our hospital from 2006 to 2016 were included in the study. Histopathological diagnosis and immunohistochemistry using a panel of antibodies (CK, CD99, CD 15, CD30, CD45, Bcl 2, anaplastic lymphoma kinase-1, CD 16, CD 57 and ki-67) were reviewed and recorded. RESULTS: Out of 153 malignant sinonasal tumors, 18 were diagnosed with lymphoma. Non-Hodgkins lymphoma constituted 88.8% of cases with the most common subtype being diffuse large B-cell lymphoma (n = 12, 66.6%). Maxillary sinus was the most frequently involved site (62%). The average age of presentation was 52 years with a slight male predominance. Computed tomography and magnetic resonance imaging scans were done in virtually all cases to assess the extent of the tumor as well as bony destruction. Average 5-year survival was 50%. Local recurrence was the most frequent cause of treatment failure. CONCLUSION: Malignant lymphomas constituted 11.7% of all malignancies of PNS. The association of diffuse large B-cell tumors with obstructive nasal mass and T-cell tumors with septal perforation, orbital extension and ophthalmological symptoms were more commonly seen.

Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase.

The crystal structure of yeast mitochondrial F(1) ATPase contains three independent copies of the complex, two of which have similar conformations while the third differs in the position of the central stalk relative to the alpha(3)beta(3) sub-assembly. All three copies display very similar asymmetric features to those observed for the bovine enzyme, but the yeast F(1) ATPase structures provide novel information. In particular, the active site that binds ADP in bovine F(1) ATPase has an ATP analog bound and therefore this structure does not represent the ADP-inhibited form. In addition, one of the complexes binds phosphate in the nucleotide-free catalytic site, and comparison with other structures provides a picture of the movement of the phosphate group during initial binding and subsequent catalysis. The shifts in position of the central stalk between two of the three copies of yeast F(1) ATPase and when these structures are compared to those of the bovine enzyme give new insight into the conformational changes that take place during rotational catalysis.

Expression of bovine F1-ATPase with functional complementation in yeast Saccharomyces cerevisiae.

The mitochondrial F(1)F(0)-ATP synthase is a multimeric enzyme complex composed of at least 16 unique peptides with an overall molecular mass of approximately 600 kDa. F(1)-ATPase is composed of alpha(3)beta(3)gammadeltaepsilon with an overall molecular mass of 370 kDa. The genes encoding bovine F(1)-ATPase have been expressed in a quintuple yeast Saccharomyces cerevisiae deletion mutant (DeltaalphaDeltabetaDeltagammaDeltadeltaDeltaepsilon). This strain expressing bovine F(1) is unable to grow on medium containing a non-fermentable carbon source (YPG), indicating that the enzyme is non-functional. However, daughter strains were easily selected for growth on YPG medium and these were evolved for improved growth on YPG medium. The evolution of the strains was presumably due to mutations, but mutations in the genes encoding the subunits of the bovine F(1)-ATPase were not required for the ability of the cell to grow on YPG medium. The bovine enzyme expressed in yeast was partially purified to a specific activity of about half of that of the enzyme purified from bovine heart mitochondria. These results indicate that the molecular machinery required for the assembly of the mitochondrial ATP synthase is conserved from bovine and yeast and suggest that yeast may be useful for the expression, mutagenesis, and analysis of the mammalian F(1)- or F(1)F(0)-ATP synthase.

Ni-chelate-affinity purification and crystallization of the yeast mitochondrial F1-ATPase.

The yeast mitochondrial ATPase has been genetically modified to include a His(6) Ni-affinity tag on the amino end of the mature beta-subunit. The modified beta-subunit is imported into the mitochondrion, properly processed to the mature form, and assembled into a mature and fully active ATP synthase. The F(1)-ATPase has been purified from submitochondrial particles after release from the membrane with chloroform, followed by Ni-chelate-affinity and gel filtration chromatography. The final enzyme is a homogeneous preparation with full activity and no apparent degradation products. This enzyme preparation has been used to obtain crystals that diffract to better than 2.8 A resolution.

Crystallization and preliminary crystallographic studies of the mitochondrial F1-ATPase from the yeast Saccharomyces cerevisiae.

A genetically modified (His6-tagged) form of the mitochondrial F1-ATPase (MW = 370 kDa) has been purified from the yeast Saccharomyces cerevisiae and crystallized in the presence of polyethelene glycol (PEG) 6000 as a precipitant, 1 mM NiCl2, 1 mM Mg AMP-PNP and 50 microM Mg ADP. X-ray diffraction data were obtained on three separate occasions using synchrotron radiation, with a progression in the quality of the diffraction data, which improved from 3.3 to 3.0 to 2.8 A. On the second occasion, the diffraction was improved by a crystal-annealing procedure. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 110.6, b = 294.2, c = 190.4 A, beta = 101.6 degrees. The asymmetric unit contains three molecules of yeast F1, with a corresponding volume per protein weight (VM) of 2.8 A3 Da(-1) and a solvent content of 55%.

SRE1 and SRE2 are two specific steroid-responsive modules of Candida drug resistance gene 1 (CDR1) promoter.

CDR1 gene encoding an ATP-driven drug extrusion pump has been implicated in the development of azole-resistance in Candida albicans. Although the upregulation of CDR1 expression by various environmental factors has been documented, the molecular mechanism underlying such process is poorly understood. We have demonstrated earlier that the CDR1 promoter encompasses a large number of cis-regulatory elements, presumably mediating its response to various drugs. In this study we have identified a novel steroid responsive region (SRR) conferring beta-oestradiol and progesterone inducibility on the CDR1 promoter. The SRR is located -696 to -521 bp upstream of the transcription start site; it is modular in nature and can confer steroid responsiveness to a heterologous promoter (ADH1) linked to a GFP reporter gene. In vitro DNase I protection analyses of SRR revealed two progesterone responsive sequences (-628 to -594 and -683 to -648) and one beta-oestradiol responsive sequence (-628 to -577), which was further corroborated by the gel mobility shift assay. Deletion analyses within the SRR further delimited these steroid responsive sequences into two distinct elements, viz. SRE1 and SRE2. While SRE1 (-677 to -648) responds only to progesterone, SRE2 (-628 to -598) responded to both progesterone and beta-oestradiol. Both SRE1 and SRE2 were specific for steroids, as they did not respond to other drugs, such as cycloheximide, miconazole and terbinafine. In silico comparison of the SRE1/2 with the promoter sequences of other MDR (CDR2 and PDR5) and non-MDR (HSP90) steroid-responsive genes revealed a similarity with respect to conservation of three 5 bp stretches (AAGAA, CCGAA and ATTGG). Taken together, we have identified a novel steroid responsive cis-regulatory sequence in the CDR1 promoter, which presumably can be instrumental in understanding the steroid response cascade in Candida albicans.

Identification of a negative regulatory element which regulates basal transcription of a multidrug resistance gene CDR1 of Candida albicans.

We have earlier shown that transcriptional activation of the Candida drug resistance gene, CDR1, is linked to various stresses wherein a proximal promoter (-345 bp from the transcription start point (TSP)) was found to be predominantly more responsive. In this study we have examined basal expression of the CDR1 proximal promoter by employing a Renilla luciferase reporter system. We observed that upon sequential deletion of the proximal promoter, there was modulation in basal reporter activity. The reporter activity was highest (2.3-fold) in NGY261 (-261 bp from TSP), and was reduced upon subsequent deletions. DNase I footprinting revealed four protected regions (W1, W2, W3 and W4) in the proximal promoter which could represent possible trans-acting factor binding sites and thus might be involved in CDR1 expression. Site-directed mutational analysis of three of these protected regions did not significantly affect the basal reporter activity, however, the mutation of W1 led to a considerable enhancement in reporter activity (approximately 4-fold) and was designated a negative regulatory element (NRE). Mutation as well as deletion of the W1 sequence in the native promoter (-1147 bp from TSP) and sequential deletion of the 5'-flanking region-harboring W1 (NRE) also resulted in enhanced promoter reporter activity. When the reporter activity of native (NPY1147) and NRE-mutated (NGYM1147) promoter integrants was monitored throughout the growth phase of Candida albicans, there was modulation in reporter activity in both integrants, but interestingly the level of basal reporter activity of the NRE-mutated promoter was always approximately 3-fold higher than that of the native promoter. UV cross-linking and affinity purification confirmed that a purified approximately 55-kDa nuclear protein specifically interacts with the NRE. Taken together, we have identified a NRE and purified its interactive protein, which may be involved in controlling basal expression of CDR1.