Molecular mechanisms associated with Fluconazole resistance in clinical Candida albicans isolates from India

Resistance to azole antifungals is a significant problem in Candida albicans. An understanding of resistance at molecular level is essential for the development of strategies to tackle resistance and rationale design of newer antifungals and target‐based molecular approaches. This study presents the first evaluation of molecular mechanisms associated with fluconazole resistance in clinical C.albicans isolates from India. Target site (ERG11) alterations were determined by DNA sequencing, whereas real‐time PCRs were performed to quantify target and efflux pump genes (CDR1, CDR2, MDR1) in 87 [Fluconazole susceptible (n = 30), susceptible‐dose dependent (n = 30) and resistant (n = 27)] C.albicans isolates. Cross‐resistance to fluconazole, ketoconazole and itraconazole was observed in 74.1% isolates. Six amino acid substitutions were identified, including 4 (E116D, F145L, E226D, I437V) previously reported ones and 2 (P406L, Q474H) new ones. CDR1 over‐expression was seen in 77.7% resistant isolates. CDR2 was exclusively expressed with CDR1 and their concomitant over‐expression was associated with azole cross‐resistance. MDR1 and ERG11 over‐expression did not seem to be associated with resistance. Our results show that drug efflux mediated by Adenosine‐5′‐triphosphate (ATP)‐binding cassette transporters, especially CDR1 is the predominant mechanism of fluconazole resistance and azole cross‐resistance in C. albicans and indicate the need for research directed towards developing strategies to tackle efflux mediated resistance to salvage azoles.     

Physiological properties of hERG 1a/1b heteromeric currents and a hERG 1b-specific mutation associated with Long-QT syndrome

Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long-QT syndrome (LQTS). Biochemical and functional studies have demonstrated that I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homooligomers of the original hERG 1a isolate. Here, we examine currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We find that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a 2-fold increase in the apparent rates of activation and recovery from inactivation, thus reducing rectification and facilitating current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the 2 channel types. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding 4-fold shift in the IC50. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients that was absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells dramatically reduced 1b protein levels. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr and enhance drug sensitivity, and represent a potential mechanism underlying inherited or acquired LQTS.     

Auditory acuity in type 2 diabetes mellitus

BACKGROUND AND OBJECTIVES:

The relationship between diabetes and hearing loss has been debated for many years. Hyperglycemia appears to have an effect on hearing loss and the proposed mechanisms are microangiopathy, neuropathy or a combination of both. The objective of this study was to evaluate a cross section of hyperglycemic subjects with age- and sex-matched normoglycemic controls with pure tone audiometry and compare the differences.

MATERIALS AND METHODS:

Forty-one type 2 diabetes mellitus subjects and 41 age- and sex-matched normoglycemic controls were subjected to a pure tone audiometric assessment followed by evaluation of their glycemic status and degree of glycemic control. The resulting data was statistically analyzed.

RESULTS:

The auditory thresholds in hyperglycemic subjects were higher in all age groups in all the frequencies suggestive of sensorineural hearing loss. The hyperglycemic subjects with poor control of their blood sugar levels (HbA1C > 8%) had elevated auditory thresholds in all the test frequencies. The fasting blood sugar level in hyperglycemic subjects showed a trend towards significant difference at higher frequencies, the postprandial blood sugar levels showed significant differences at higher frequencies. There was no effect of duration of diabetes mellitus on the hearing thresholds in hyperglycemic subjects.

CONCLUSION:

Subjects with hyperglycemia have a sensorineural hearing loss when evaluated with a pure tone audiometer in all frequencies than a normoglycemic control group. The study showed that post prandial blood sugar levels and HbA1C levels had a direct bearing on the auditory acuity of the hyperglycemic subjects.     

In vitro Comparison of Adipogenic Differentiation in Human Adipose-Derived Stem Cells Cultured with Collagen Gel and Platelet-Rich Fibrin

Background: Adipose-derived stem cells (ADSCs) are the most preferred cell type, based on their phenotypic characteristics, plasticity, and favorable immunological properties for applications in soft-tissue augmentation. Hence, the present in vitro study was aimed to evaluate the adipogenic differentiation potential of human ADSCs upon culturing individually with collagen gel and platelet-rich fibrin (PRF). Materials and methods: The collected lipoaspirate was used for establishing ADSCs using enzymatic digestion method. Then, the cells were analyzed for their morphology, viability, proliferation rate, population doubling time (PDT), colony-forming ability, cell surface markers expression, and osteogenic differentiation as biological properties. Further, ADSCs were evaluated for their adipogenicity using induction media alone, and by culturing with collagen gel and PRF individually for prospective tissue augmentation. Results: ADSCs were successfully established in vitro and exhibited a fibroblast-like morphology throughout the culture period. Cells had higher viability, proliferation potential and showed their ability to form colonies. The positive expression of cell surface markers and osteogenic ability confirmed the potency of ADSCs. The ADSCs cultured on collagen gel and PRF, individually, showed higher number of differentiated adipocytes than ADSCs grown with adipogenic induction medium alone. Conclusion: The extent of lipid accumulation by ADSCs was slightly higher when cultured on collagen gel than on PRF. Additional experiments are required to confirm better suitability of scaffold materials for soft-tissue regeneration.     

Genomic Analysis Reveals Distinct Subtypes in Two Rare Cases of Primary Ovarian Lymphoma

Primary (localized) non-Hodgkin lymphoma (NHL) of the ovary is extremely rare; only a few cases have been reported in the literature. We report two cases of primary ovarian lymphoma (POL), one involving bilateral ovaries in a 15-year-old girl and other involving one ovary in a 5-year-old girl. This report describes detailed clinical, histopathological, and imaging findings, along with the review of literature of primary diffuse large B-cell lymphoma (DLBCL) arising from an ovary. In addition, we describe findings of targeted capture panel sequencing on both tumors and identify the major genetic mutations that are recurrently mutated in pan-cancers. Compared to the genomic mutation features of major subtypes of DLBCL, we distinguish that each POL belongs to distinctive subtypes, GCB (germinal center B-cell subtype) DLBCL and ABC (activated B-cell subtype) DLBCL, respectively. The findings from the genomic analysis may help to understand the pathogenesis of POL and to guide potential targeted therapy in the future.     

The spectrum of neurological manifestations and genotype–phenotype correlation in Indian children with Gaucher disease

Gaucher disease (GD), one of the most frequent autosomal recessive lysosomal storage disorders, occurs due to bi-allelic pathogenic variants in the GBA1. Worldwide, the c.1448T>C (L483P) homozygous pathogenic variant is reported to be associated with neurological GD phenotype. Clinical distinction between GD1 and GD3 may be challenging due to subtle neurological features. Objective methods to evaluate neurological signs and saccades may help in early diagnosis. This study was conducted to assess the neurological phenotype, and its severity using a modified severity scoring tool (mSST), and the genotype–phenotype correlation. A total of 45 children aged 2 years 6 months to 15 years with a confirmed enzymatic and molecular diagnosis of GD with or without therapy were recruited. mSST tool was used to assess the severity of the neurological phenotype. A digital eye movement tracker (View Point Tracker) was used to assess eye movements. Clinical and genetic findings were analyzed. Out of 45 patients, 39 (86.7%) had at least one neurological phenotype detected using the mSST tool, with impairment of cognitive function (68.8%, 31/45) being the commonest feature. Thirty-two of 45 (71%) were assessed for saccadic eye movements using the eye tracker. Of these, 62.5% (20/32) had absent saccades. Four children (8.9%, 4/32) without clinical oculomotor apraxia had absent saccades on the viewpoint eye tracker. Overall, 77.7% (35/45), had homozygosity for c.1448T>C in GBA1 of which 91.4% (32/35) had neurological manifestations. Other alleles associated with neurological phenotype included c.1603C>T(p.R535C), c.1184C>T (p.S395F), c.115+1G>A (g.4234G>A), c.260G>A (p.R87Q) and c.1352A>G (p.Y451C). To conclude, in India, the c.1448T>C pathogenic variant in GBA1 is the commonest  and is associated with neurological phenotype of GD. Therefore, every patient of GD should be assessed using the mSST scoring tool for an early pick up of neurological features. The routine use of a viewpoint eye tracker in children with GD would be useful for early recognition of saccadic abnormalities.