Rare case of early neonatal sepsis caused by Candida krusei successfully treated with voriconazole.

This study reports a case of multidrug resistant Candida krusei as the cause of early neonatal sepsis in a term small-for-gestational age neonate weighing 1680 g that successfully responded to voriconazole therapy. Both blood culture and urine culture of the neonate sent on day 4 and day 8 respectively showed Gram positive oval budding yeast cells on Gram staining which was confirmed as C. krusei susceptible only to voriconazole by Vitek 2 Compact (Biomérieux, France) automated system. Voriconazole was given for fourteen days leading to good clinical response with microbiological clearance of fungus from blood and no side-effects.

Clinico-Pathological Factors Determining Recurrence of Phyllodes Tumors of the Breast: The 25-Year Experience at a Tertiary Cancer Centre.

BACKGROUND: Phyllodes tumors (PTs) of the breast are rare fibroepithelial tumors that are generally more prone to recurrence. AIMS AND OBJECTIVES: This study aimed to assess the clinicopathological features, diagnostic modalities, and therapeutic interventions, along with their respective outcomes, to identify the factors associated with a recurrence of PTs of the breast. METHODOLOGY: A retrospective cohort and observational study was conducted, which entailed analyzing the clinicopathological data of patients who were previously diagnosed or presented with PTs of the breast between 1996 and 2021. Data included the total number of patients diagnosed with PTs of the breast and their ages, tumor grade on initial biopsy, tumor location (left or right breast), tumor size, therapeutic interventions carried out (including surgery-either mastectomy or lumpectomy-and adjuvant radiotherapy), final tumor grade, recurrence status, type of recurrence, and time to recurrence. RESULTS: We analyzed data on a total of 87 patients who were pathologically proven to have PTs, and 46 patients (52.87%) were found to have recurrences. All patients were female, with a mean age at diagnosis of 39 years (range 15-70). Patients aged <40 years had the highest incidence of recurrence, with a rate of 54.35% (n = 25/46), followed by patients aged >40 years, with a rate of recurrence of 45.65% (n = 21/46). A total of 55.4% of patients presented with primary PTs and 44.6% had recurrent PTs at presentation. The average time to local recurrence (LR) from the completion of treatment was 13.8 months, whereas for systemic recurrence (SR), it was 15.29 months. Surgery (mastectomy/lumpectomy) was the major determinant for local recurrence (p < 0.05). CONCLUSION: Patients who received adjuvant radiotherapy (RT) had a minimal recurrence of PTs. Patients who were found to have a malignant biopsy on initial diagnosis (triple assessment) had a higher incidence of PTs and were more prone to SR than LR. Surgery was a determining factor in the increased rate of LR, with lumpectomy associated with a higher incidence of LR than mastectomy.

Loss of Drosophila Clueless differentially affects the mitochondrial proteome compared to loss of Sod2 and Pink1.

Mitochondria contain their own DNA, mitochondrial DNA, which encodes thirteen proteins. However, mitochondria require thousands of proteins encoded in the nucleus to carry out their many functions. Identifying the definitive mitochondrial proteome has been challenging as methods isolating mitochondrial proteins differ and different tissues and organisms may have specialized proteomes. Mitochondrial diseases arising from single gene mutations in nucleus encoded genes could affect the mitochondrial proteome, but deciphering which effects are due to loss of specific pathways or to accumulated general mitochondrial damage is difficult. To identify specific versus general effects, we have taken advantage of mutations in three Drosophila genes, clueless, Sod2, and Pink1, which are required for mitochondrial function through different pathways. We measured changes in each mutant's mitochondrial proteome using quantitative tandem mass tag mass spectrometry. Our analysis identified protein classes that are unique to each mutant and those shared between them, suggesting that some changes in the mitochondrial proteome are due to general mitochondrial damage whereas others are gene specific. For example, clueless mutants had the greatest number of less and more abundant mitochondrial proteins whereas loss of all three genes increased stress and metabolism proteins. This study is the first to directly compare in vivo steady state levels of mitochondrial proteins by examining loss of three pathways critical for mitochondrial function. These data could be useful to understand disease etiology, and how mutations in genes critical for mitochondrial function cause specific mitochondrial proteomic changes as opposed to changes due to generalized mitochondrial damage.

Reduction of Drosophila Mitochondrial RNase P in Skeletal and Heart Muscle Causes Muscle Degeneration, Cardiomyopathy, and Heart Arrhythmia.

In this study, we examine the cause and progression of mitochondrial diseases linked to the loss of mtRNase P, a three-protein complex responsible for processing and cleaving mitochondrial transfer RNAs (tRNA) from their nascent transcripts. When mtRNase P function is missing, mature mitochondrial tRNA levels are decreased, resulting in mitochondrial dysfunction. mtRNase P is composed of Mitochondrial RNase P Protein (MRPP) 1, 2, and 3. MRPP1 and 2 have their own enzymatic activity separate from MRPP3, which is the endonuclease responsible for cleaving tRNA. Human mutations in all subunits cause mitochondrial disease. The loss of mitochondrial function can cause devastating, often multisystemic failures. When mitochondria do not provide enough energy and metabolites, the result can be skeletal muscle weakness, cardiomyopathy, and heart arrhythmias. These symptoms are complex and often difficult to interpret, making disease models useful for diagnosing disease onset and progression. Previously, we identified Drosophila orthologs of each mtRNase P subunit (Roswell/MRPP1, Scully/MRPP2, Mulder/MRPP3) and found that the loss of each subunit causes lethality and decreased mitochondrial tRNA processing in vivo. Here, we use Drosophila to model mtRNase P mitochondrial diseases by reducing the level of each subunit in skeletal and heart muscle using tissue-specific RNAi knockdown. We find that mtRNase P reduction in skeletal muscle decreases adult eclosion and causes reduced muscle mass and function. Adult flies exhibit significant age-progressive locomotor defects. Cardiac-specific mtRNase P knockdowns reduce fly lifespan for Roswell and Scully, but not Mulder. Using intravital imaging, we find that adult hearts have impaired contractility and exhibit substantial arrhythmia. This occurs for roswell and mulder knockdowns, but with little effect for scully. The phenotypes shown here are similar to those exhibited by patients with mitochondrial disease, including disease caused by mutations in MRPP1 and 2. These findings also suggest that skeletal and cardiac deficiencies induced by mtRNase P loss are differentially affected by the three subunits. These differences could have implications for disease progression in skeletal and heart muscle and shed light on how the enzyme complex functions in different tissues.

Loss of Individual Mitochondrial Ribonuclease P Complex Proteins Differentially Affects Mitochondrial tRNA Processing In Vivo.

Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5'-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.

Determinants of lymph node status in women with breast cancer: A hospital based study from eastern India.

BACKGROUND & OBJECTIVES: Number of metastatic lymph nodes has a strong prognostic value in the course of breast cancer treatment, morbidity and mortality. This study was undertaken to determine the association between axillary lymph node metastasis and several variables such as age, tumour size, grade, lymphovascular invasion, oestrogen and progesterone receptor expression and HER2/neu status in patients with breast cancer. METHODS: In this study 426 (with complete information on study variables) patients with breast cancer on treatment during March 2010 to December 2013, were analyzed. TNM (tumour node matastasis) staging was evaluated. The histological grading of tumours was done according to modified Bloom-Richardson Grading System. The immunophenotype of the tumour was determined as the expression of oestrogen (ER) and progesterone (PR) receptors and Her0 2/neu status. Univariate and multivariate analyses were carried out to determine the independent predictors of metastatic lymph node. RESULTS: Among the studied patients, 44.36 per cent (189 of 426) of the patients had nodal metastases. t0 umour histology, tumour grade, size and lympho-vascular invasion were related with node positivity. On univariate analysis, age, menopause, hormone receptor status did not relate with the node metastasis. Age, tumour grade, tumour size, lympho-vascular invasion and HER2/neu expression was likely to be associated with the number of lymph node metastasis. INTERPRETATION & CONCLUSIONS: The lymph node status was associated with clinical stage, tumour grade, tumour histology and HER2/neu status. t0 hese factors may be used for better management of such patients.

Loss of the mitochondrial protein-only ribonuclease P complex causes aberrant tRNA processing and lethality in Drosophila.

Proteins encoded by mitochondrial DNA are translated using mitochondrially encoded tRNAs and rRNAs. As with nuclear encoded tRNAs, mitochondrial tRNAs must be processed to become fully functional. The mitochondrial form of ribonuclease P (mt:RNase P) is responsible for 5'-end maturation and is comprised of three proteins; mitochondrial RNase P protein (MRPP) 1 and 2 together with proteinaceous RNase P (PRORP). However, its mechanism and impact on development is not yet known. Using homology searches, we have identified the three proteins composing Drosophila mt:RNase P: Mulder (PRORP), Scully (MRPP2) and Roswell (MRPP1). Here, we show that each protein is essential and localizes with mitochondria. Furthermore, reducing levels of each causes mitochondrial deficits, which appear to be due at least in part to defective mitochondrial tRNA processing. Overexpressing two members of the complex, Mulder and Roswell, is also lethal, and in the case of Mulder, causes abnormal mitochondrial morphology. These data are the first evidence that defective mt:RNase P causes mitochondrial dysfunction, lethality and aberrant mitochondrial tRNA processing in vivo, underscoring its physiological importance. This in vivo mt:RNase P model will advance our understanding of how loss of mitochondrial tRNA processing causes tissue failure, an important aspect of human mitochondrial disease.

Clueless is a conserved ribonucleoprotein that binds the ribosome at the mitochondrial outer membrane.

Mitochondrial function is tied to the nucleus, in that hundreds of proteins encoded by nuclear genes must be imported into mitochondria. While post-translational import is fairly well understood, emerging evidence supports that mitochondrial site-specific import, or co-translational import, also occurs. However, the mechanism and the extent to which it is used are not fully understood. We have previously shown Clueless (Clu), a conserved multi-domain protein, associates with mitochondrial outer membrane proteins, including Translocase of outer membrane 20, and genetically and physically interacts with the PINK1-Parkin pathway. The human ortholog of Clu, Cluh, was shown to bind nuclear-encoded mitochondrially destined mRNAs. Here we identify the conserved tetratricopeptide domain of Clu as predominantly responsible for binding mRNA. In addition, we show Clu interacts with the ribosome at the mitochondrial outer membrane. Taken together, these data support a model whereby Clu binds to and mitochondrially targets mRNAs to facilitate mRNA localization to the outer mitochondrial membrane, potentially for site-specific or co-translational import. This role may link the presence of efficient mitochondrial protein import to mitochondrial quality control through the PINK1-Parkin pathway.

Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila.

Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease (PD). However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1) and Parkin (Park), either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. Here, we show that Drosophila Clueless (Clu), a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM) 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previously, we found that clu genetically interacts with park in Drosophila female germ cells. Here, we show that clu also genetically interacts with PINK1, and our epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control.

Optomotor-blind in the development of the Drosophila HS and VS lobula plate tangential cells.

The horizontal system and vertical system cells of the dipteran optic lobes are well understood regarding their physiology and role in visually guided behavior. Little is known, however, about their development. Drosophila optomotor-blind (omb) is required for the development of the HS/VS cells which are lacking in the adult brain of the In(1)omb[H31] regulatory mutant. We have analyzed the omb regulatory region, required for HS/VS development, for enhancers active in the central nervous system. A 1-kb fragment, ombJb, was identified 114 kb downstream of the omb transcription start site, that could drive expression in much of the presumptive embryonic optic lobe anlage. Expression in these cells is lost in In(1)omb[H31] suggesting that they contain the HS/VS precursor cell(s). We used Laser ablation in the embryonic CNS in order to localize the position of the HS/VS precursor cell(s) in this tissue. An omb-Gal4 enhancer trap line, which showed activity in the optic lobe anlage in a pattern similar to ombJb enhancer, was used to drive GFP expression, thus allowing to focus the Laser beam to the relevant area. We identified a small region in the embryonic brain from which the HS/VS cells are likely to develop. Omb encodes a transcription factor of the T-box family. Since loss of omb disrupts HS/VS cell development, we assume that HS/VS ontogeny is controlled by Omb target genes. As a first step toward their identification, we characterized the Omb DNA-binding specificity.

Drosophila clueless is highly expressed in larval neuroblasts, affects mitochondrial localization and suppresses mitochondrial oxidative damage.

Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu) has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, in this study we have identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. We show while mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, overall brain morphology appears to be normal. This is explained by our observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu's specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus Clu's role in mitochondrial function is not critical during larval development, but is important for pupae and adults.

Odd paired transcriptional activation of decapentaplegic in the Drosophila eye/antennal disc is cell autonomous but indirect.

The gene odd paired (opa), a Drosophila homolog of the Zinc finger protein of the cerebellum (Zic) family of mammalian transcription factors, plays roles in embryonic segmentation and development of the adult head. We have determined the preferred DNA binding sequence of Opa by SELEX and shown that it is necessary and sufficient to activate transcription of reporter gene constructs under Opa control in transgenic flies. We have found a related sequence in the enhancer region of an opa-responsive gene, sloppy paired 1. This site also responds to Opa in reporter constructs in vivo. However, nucleotide alterations that abolish the ability of Opa to bind this site in vitro have no effect on the ability of Opa to activate expression from constructs bearing these mutations in vivo. These data suggest that while Opa can function in vivo as a sequence specific transcriptional regulator, it does not require DNA binding for transcriptional activation.

Null mutations in Drosophila Optomotor-blind affect T-domain residues conserved in all Tbx proteins.

The T-box transcription factors TBX2 and TBX3 are overexpressed in many human cancers raising the need for a thorough understanding of the cellular function of these proteins. In Drosophila, there is one corresponding ortholog, Optomotor-blind (Omb). Currently, only two missense mutations are known for the two human proteins. Making use of the developmental defects caused by inactivation of omb, we have isolated and molecularly characterized four new omb mutations, three of them are missense mutations of amino acids fully conserved in all Tbx proteins. We interpret the functional defects in the framework of the known structure of the human TBX3 protein and provide evidence for loss of Omb DNA-binding activity in all three newly identified missense mutations.