Rapid detection of Mucorales in human blood and urine samples by functionalized Heusler magnetic nanoparticle assisted customized loop-mediated isothermal amplification.

Mucormycosis is a rare disease with scarce diagnostic methods for early intervention. Available strategies employing direct microscopy using calcofluor white-KOH, culture, radiologic, and histopathologic testing often are time-intensive and demand intricate protocols. Nucleic Acid Amplification Test holds promise due to its high sensitivity combined with rapid detection. Loop-mediated isothermal amplification (LAMP) based detection offers an ultrasensitive technique that does not require complicated thermocyclers like in polymerase chain reaction, offering a straightforward means for improving diagnoses as a near-point-of-care test. The study introduces a novel magnetic nanoparticle-based LAMP assay for carryover contaminant capture to reduce false positives. Solving the main drawback of LAMP-based diagnosis techniques. The assay targets the cotH gene, which is invariably specific to Mucorales. The assay was tested with various species of Mucorales, and the limit of detections for Rhizopus microsporus, Lichtheimia corymbifera, Rhizopus arrhizus, Rhizopus homothallicus, and Cunninghamella bertholletiae were 1 fg, 1 fg, 0.1 pg, 0.1 pg, and 0.01 ng, respectively. This was followed by a clinical blindfolded study using whole blood and urine samples from 30 patients diagnosed with Mucormycosis. The assay has a high degree of repeatability and had an overall sensitivity of > 83%. Early Mucormycosis detection is crucial, as current lab tests from blood and urine lack sensitivity and take days for confirmation despite rapid progression and severe complications. Our developed technique enables the confirmation of Mucormycosis infection in < 45 min, focusing specifically on the RT-LAMP process. Consequently, this research offers a viable technique for quickly identifying Mucormycosis from isolated DNA of blood and urine samples instead of invasive tissue samples.

Mucormycosis is a challenging disease to diagnose early. This study introduces a sensitive and rapid diagnostic approach using Loop-mediated isothermal amplification technology. Testing blood and urine samples from 30 patients revealed promising sensitivity and repeatability, indicating its potential for non-invasive diagnosis.

Prostate Cancer Progression Relies on the Mitotic Kinase Citron Kinase.

Prostate cancer remains the second leading cause of cancer death in men in Western cultures. A deeper understanding of the mechanisms by which prostate cancer cells divide to support tumor growth could help devise strategies to overcome treatment resistance and improve survival. Here, we identified that the mitotic AGC family protein kinase citron kinase (CIT) is a pivotal regulator of prostate cancer growth that mediates prostate cancer cell interphase progression. Increased CIT expression correlated with prostate cancer growth induction and aggressive prostate cancer progression, and CIT was overexpressed in prostate cancer compared with benign prostate tissue. CIT overexpression was controlled by an E2F2-Skp2-p27 signaling axis and conferred resistance to androgen-targeted treatment strategies. The effects of CIT relied entirely on its kinase activity. Conversely, CIT silencing inhibited the growth of cell lines and xenografts representing different stages of prostate cancer progression and treatment resistance but did not affect benign epithelial prostate cells or nonprostatic normal cells, indicating a potential therapeutic window for CIT inhibition. CIT kinase activity was identified as druggable and was potently inhibited by the multikinase inhibitor OTS-167, which decreased the proliferation of treatment-resistant prostate cancer cells and patient-derived organoids. Isolation of the in vivo CIT substrates identified proteins involved in diverse cellular functions ranging from proliferation to alternative splicing events that are enriched in treatment-resistant prostate cancer. These findings provide insights into the regulation of aggressive prostate cancer cell behavior by CIT and identify CIT as a functionally diverse and druggable driver of prostate cancer progression. SIGNIFICANCE: The poorly characterized protein kinase citron kinase is a therapeutic target in prostate cancer that drives tumor growth by regulating diverse substrates, which control several hallmarks of aggressive prostate cancer progression. See related commentary by Mishra et al., p. 4008.

Impact of anxiety and depression on disease activity and quality of life in patients with lupus nephritis.

Background of the Study: Lupus is an autoimmune disease that affects multiple body systems and requires long-term treatment. The multisystem effect of this disease and long treatment may cause anxiety and depression in patients with lupus nephritis (LN) and ultimately deteriorate their quality of life and also affects the activity of the disease. Aim of the Study: This study aims to assess anxiety, depression, and quality of life in patients with LN and their relationship with disease activity. Material and Methods: A descriptive cross-sectional study was conducted to assess anxiety, depression, and quality of life among patients with LN. A total enumerative technique was used for the recruitment of 100 patients and data collected using standardized tools were analyzed. Results: The results of the study showed that the majority of patients (60.0%) with LN had moderate anxiety and most of them (61.0%) had moderate depression that affected their quality of life and impacted the disease activity index in lupus. Conclusion: LN patients experience significant levels of anxiety and depression, which deteriorates their quality of life and negatively impacts disease activity. Active surveillance for these conditions and early diagnosis might help in the improvement of health-related outcomes in such patients.

Factors affecting quality of daytime and nighttime sleep among dialysis patients: A single center experience.

BACKGROUND: Hemodialysis is the most common treatment modality for patients with chronic kidney disease (CKD). Excessive daytime sleepiness and poor nighttime sleep is a common problem among these patients. Patients on maintenance hemodialysis (MHD) are regularly exposed to impaired fluid balance, which may cause overhydration of varying degree. However, the role of hydration status in sleep quality has not been explored in Indian setting. Hence, this study was undertaken to assess the factors affecting sleep quality among patients on MHD in a tertiary care hospital. MATERIAL AND METHODS: Patients (N = 55) were enrolled if they aged above18 years, on MHD for at least 3 months, and gave consent. The daytime sleep quality was assessed using Epworth Sleepiness Scale (ESS) and Insomnia Severity Index (ISI). The data were analyzed using SPSS version 20 and STATA software. RESULTS: The mean age of the patients was 40.4 ± 14.7 years. The prevalence rate of predialysis fluid overload was 85.4%. The median ESS score was 7 and ISI score was 3 indicating normal daytime sleep and not significant insomnia. Multivariate regression with variables adjustment showed that interdialytic weight gain (P = 0.33), tingling sensation (P = 0.36) and numbness (P = 0.35) were significant predictive factors for quality of sleep. CONCLUSION: The major factors affecting sleep quality were numbness, tingling sensation, and interdialytic weight gain. Fluid overload did not play any role in sleep quality. Another study may be carried out on assessment of pattern, duration, quality of sleep in multiple dialysis sessions, and effect of optimizing fluid status on the sleep parameters.

Analyzing the Androgen Receptor Interactome in Prostate Cancer: Implications for Therapeutic Intervention.

The androgen receptor (AR) is a member of the ligand-activated nuclear receptor family of transcription factors. AR's transactivation activity is turned on by the binding of androgens, the male sex steroid hormones. AR is critical for the development and maintenance of the male phenotype but has been recognized to also play an important role in human diseases. Most notably, AR is a major driver of prostate cancer (CaP) progression, which remains the second leading cause of cancer deaths in American men. Androgen deprivation therapies (ADTs) that interfere with interactions between AR and its activating androgen ligands have been the mainstay for treatment of metastatic CaP. Although ADTs are effective and induce remissions, eventually they fail, while the growth of the majority of ADT-resistant CaPs remains under AR's control. Alternative approaches to inhibit AR activity and bypass resistance to ADT are being sought, such as preventing the interaction between AR and its cofactors and coregulators that is needed to execute AR-dependent transcription. For such strategies to be efficient, the 3D conformation of AR complexes needs to be well-understood and AR-regulator interaction sites resolved. Here, we review current insights into these 3D structures and the protein interaction sites in AR transcriptional complexes. We focus on methods and technological approaches used to identify AR interactors and discuss challenges and limitations that need to be overcome for efficient therapeutic AR complex disruption.

Breast tumor-on-chip models: From disease modeling to personalized drug screening.

Breast cancer is one of the leading causes of mortality worldwide being the most common cancer among women. Despite the significant progress obtained during the past years in the understanding of breast cancer pathophysiology, women continue to die from it. Novel tools and technologies are needed to develop better diagnostic and therapeutic approaches, and to better understand the molecular and cellular players involved in the progression of this disease. Typical methods employed by the pharmaceutical industry and laboratories to investigate breast cancer etiology and evaluate the efficiency of new therapeutic compounds are still based on traditional tissue culture flasks and animal models, which have certain limitations. Recently, tumor-on-chip technology emerged as a new generation of in vitro disease model to investigate the physiopathology of tumors and predict the efficiency of drugs in a native-like microenvironment. These microfluidic systems reproduce the functional units and composition of human organs and tissues, and importantly, the rheological properties of the native scenario, enabling precise control over fluid flow or local gradients. Herein, we review the most recent works related to breast tumor-on-chip for disease modeling and drug screening applications. Finally, we critically discuss the future applications of this emerging technology in breast cancer therapeutics and drug development.

Functionalized Co2FeAl Nanoparticles for Detection of SARS CoV-2 Based on Reverse Transcriptase Loop-Mediated Isothermal Amplification.

Loop-mediated isothermal amplification (LAMP) is a sensitive, efficient, and rapid nucleic acid amplification technique resulting in a large number of amplicons; however, it suffers from a high incidence of false positives due to carry-over and aerosol. Herein, we report a 10 min nano-capture system that is used in conjunction with a modified reverse transcriptase-LAMP (RT-LAMP) assay for the accurate detection of SARS CoV-2 virus. The nano-capture system employs in-house-designed probe-functionalized magnetic nanoparticles Co2FeAl (cobalt-based Heusler alloy) for efficient capture of contaminating amplicons from the reaction mixture preceding RT-LAMP. The nano-cleaned RT-LAMP assay along with engineered primers successfully detected the presence of 10 copies of SARS CoV-2 virus while completely eliminating the incidence of false positives. The presented contaminant-capture method has been compared with other approaches for elimination of contaminants and was found to be more effective. The insight brought in this work is the design of a rapid nano-capture system that hybridizes with contaminating amplicons (carry-over) with high specificity to enable easy removal from the assay for elimination of false positives. The method has been proven to be successful for RT-LAMP assays in the rapid and highly specific detection of SARS CoV-2, which is currently a major challenge for global health. To the best of our knowledge, this is the first work involving a nano-based cleaning strategy for reliable and rapid diagnosis using isothermal amplification approaches.

Erratum: Majumder, S. et al. Zinc Oxide Nanoparticles Functionalized on Hydrogel Grafted Silk Fibroin Fabrics as Efficient Composite Dressing. Biomolecules 2020, 10, 710.

The authors wish to make the following correction to this paper [...].

Zinc Oxide Nanoparticles Functionalized on Hydrogel Grafted Silk Fibroin Fabrics as Efficient Composite Dressing.

Recent advances in woundcare is targeted towards developing active-dressings, where multiple components are combined to provide a suitable environment for rapid healing. The aim of the present research is to study the preparation of biomimic composite wound dressings by the grafting of hydrogel on silk fibroin fabric. The swelling ability of hydrogel grafted silk fibroin fabric was optimized by changing the initiator concentration. In order to impart antimicrobial properties, these dressing are further coated sono-chemically with zinc oxide nanoparticles. The water vapor transmission rate of the prepared samples was measured. The conformation of silk fibroin proteins after grafting with hydrogel was also confirmed using Fourier Transform Infrared Spectroscopy (FTIR). The morphology of the zinc oxide-coated silk fibroin fabric and hydrogel-coated silk fibroin was studied using Scanning Electron Microscope (SEM). The antimicrobial activity of the zinc oxide-coated samples was studied against E coli. The cytocompatibility of the prepared dressing materials were evaluated using L929 fibroblast cells. MTT assay and phase contrast microscopic studies showed that the adherence, growth, and proliferation of the L929 fibroblast cells that were seeded on zinc oxide nanoparticles on the functionalized hydrogel-coated silk fibroin dressing was significantly higher than that of pure silk fibroin due to the highly porous, bio-mimic structure that allowed ease of passage of nutrients, growth factors, metabolites, and the exchange of gases which is beneficial for successful regeneration of damaged tissues. The expression of TNF-α and IL-2 were not significantly higher than that of control. The proposed composite dressing would be a promising material for wound dressing and regenerative medicine but in order to prove the efficacy of these materials, more in vivo experiments and clinical tests are required to be conducted in future.

Downregulation of peripheral PTGS2/COX-2 in response to valproate treatment in patients with epilepsy.

Antiepileptic drug therapy has significant inter-patient variability in response towards it. The current study aims to understand this variability at the molecular level using microarray-based analysis of peripheral blood gene expression profiles of patients receiving valproate (VA) monotherapy. Only 10 unique genes were found to be differentially expressed in VA responders (n = 15) and 6 genes in the non-responders (n = 8) (fold-change >2, p < 0.05). PTGS2 which encodes cyclooxygenase-2, COX-2, showed downregulation in the responders compared to the non-responders. PTGS2/COX-2 mRNA profiles in the two groups corresponded to their plasma profiles of the COX-2 product, prostaglandin E2 (PGE2). Since COX-2 is believed to regulate P-glycoprotein (P-gp), a multidrug efflux transporter over-expressed at the blood-brain barrier (BBB) in drug-resistant epilepsy, the pathway connecting COX-2 and P-gp was further explored in vitro. Investigation of the effect of VA upon the brain endothelial cells (hCMEC/D3) in hyperexcitatory conditions confirmed suppression of COX-2-dependent P-gp upregulation by VA. Our findings suggest that COX-2 downregulation by VA may suppress seizure-mediated P-gp upregulation at the BBB leading to enhanced drug delivery to the brain in the responders. Our work provides insight into the association of peripheral PTGS2/COX-2 expression with VA efficacy and the role of COX-2 as a potential therapeutic target for developing efficacious antiepileptic treatment.

Role of Cellular Retention and Intracellular State in Controlling Gene Delivery Efficiency of Multiple Nonviral Carriers.

Nonviral gene delivery has seen major progress in the last two decades owing to facile synthesis, low toxicity, and ease of modification of nanocarriers that take nucleic acids to cells and tissues. Gene delivery nanocomplexes need to reach the target locations in significant amounts by overcoming multiple barriers. While the importance of nanocomplex stability, cellular uptake, intracellular trafficking, and nuclear localization has been studied extensively, the role of cellular retention and recycling of these nanocomplexes is less understood in the context of gene delivery. In this study, we used different DNA carriers and made efforts to understand the role played by cellular retention in determining their gene delivery efficiency across multiple cell lines. In addition, we also analyzed whether state of complexation and localization of the nanocomplexes play a role in conjunction with cellular retention. We observed higher transfection efficiencies for nanocomplexes showing better retention, lower unpackaging, and low recycling. Our data also suggests that nanocomplexes made of peptides with terminal cysteine modification show enhanced retention and transfection efficiency compared to their counterparts with no terminal cysteine. Overall, the work highlights myriad of factors to be considered for improving gene delivery efficiency of nanocomplexes.

Cyclooxygenase-2 (COX-2) inhibitors: future therapeutic strategies for epilepsy management.

Epilepsy, a common multifactorial neurological disease, affects about 69 million people worldwide constituting nearly 1% of the world population. Despite decades of extensive research on understanding its underlying mechanism and developing the pharmacological treatment, very little is known about the biological alterations leading to epileptogenesis. Due to this gap, the currently available antiepileptic drug therapy is symptomatic in nature and is ineffective in 30% of the cases. Mounting evidences revealed the pathophysiological role of neuroinflammation in epilepsy which has shifted the focus of epilepsy researchers towards the development of neuroinflammation-targeted therapeutics for epilepsy management. Markedly increased expression of key inflammatory mediators in the brain and blood-brain barrier may affect neuronal function and excitability and thus may increase seizure susceptibility in preclinical and clinical settings. Cyclooxygenase-2 (COX-2), an enzyme synthesizing the proinflammatory mediators, prostaglandins, has widely been reported to be induced during seizures and is considered to be a potential neurotherapeutic target for epilepsy management. However, the efficacy of such therapy involving COX-2 inhibition depends on various factors viz., therapeutic dose, time of administration, treatment duration, and selectivity of COX-2 inhibitors. This article reviews the preclinical and clinical evidences supporting the role of COX-2 in seizure-associated neuroinflammation in epilepsy and the potential clinical use of COX-2 inhibitors as a future strategy for epilepsy treatment.

Exocytosis - a putative road-block in nanoparticle and nanocomplex mediated gene delivery.

Gene and drug delivery mediated by nanostructures has seen tremendous growth over the last decade. However, the efficiency of these delivery approaches needs to be improved for better effects. Amongst various factors, cellular retention is expected to play a critical role. Nanoparticles and nanocomplexes internalized by the cells can be recycled back to the outside through the process of exocytosis. Although it sounds reasonable that the efficiency of these delivery systems should not only depend on their cellular uptake but also on the ability of cells to retain them, the process of cellular retention and exocytosis is relatively less studied in the literature. In the context of gene delivery, both inorganic nanoparticles and organic nanocomplexes are used, but there is limited information on how these nanoparticles and nanocomplexes are recycled and what could be the possible effect of such recycling on the efficiency of these delivery vectors. In this review we try to summarize the existing literature in this area, putative mechanisms involved in recycling of the nanoparticles, methods used to quantify exocytosis and factors affecting exocytosis. The possibility of enhancing cellular retention by blocking recycling pathways as well as the in vivo implications is also discussed here.

Admission Vitamin D status does not predict outcome of critically ill patients on mechanical ventilation: An observational pilot study.

BACKGROUND AND AIMS: Effect of serum 25-hydroxy vitamin D (25[OH] D) levels on the recovery of critically ill mechanically ventilated patients is unclear. Hence, this study assessed 25(OH)D levels of critically ill patients on mechanical ventilation at the time of admission to the Intensive Care Unit (ICU) and its relationship with clinical outcome. METHODS: In this prospective observational pilot study, forty adult patients receiving mechanical ventilation in the ICU were included. Serum 25(OH)D was assessed within 24 h of admission. Primary outcome was 30-day mortality and secondary outcomes were days on mechanical ventilation, ICU-length of stay (ICU-LOS), days to reach spontaneous breathing trial (SBT), requirement of advanced care modality and complications. RESULTS: Seventy-five percent patients had low serum 25(OH)D (65% deficient and 10% insufficient). Between patients with low and normal vitamin D, there was no significant difference in 30-day mortality (10% vs. 16.7%; P = 0.81), days on mechanical ventilation (16.2 ± 8.9 vs. 19.9 ± 8.4; P = 0.23), ICU-length of stay (18.7 ± 8.5 vs. 23.3 ± 11.4; P = 0.28), days to reach SBT (11.5 (0-20) vs. 21 (8-30); P = 0.78), complications developed during ICU stay (P = 0.60) and need for advanced care modalities (P = 0.72). CONCLUSION: Low Vitamin D level at admission did not affect 30-day mortality of critically ill patients on mechanical ventilation.

Molecular characterization and expression of a novel alcohol oxidase from Aspergillus terreus MTCC6324.

The alcohol oxidase (AOx) cDNA from Aspergillus terreus MTCC6324 with an open reading frame (ORF) of 2001 bp was constructed from n-hexadecane induced cells and expressed in Escherichia coli with a yield of ∼4.2 mg protein g-1 wet cell. The deduced amino acid sequences of recombinant rAOx showed maximum structural homology with the chain B of aryl AOx from Pleurotus eryngii. A functionally active AOx was achieved by incubating the apo-AOx with flavin adenine dinucleotide (FAD) for ∼80 h at 16°C and pH 9.0. The isoelectric point and mass of the apo-AOx were found to be 6.5±0.1 and ∼74 kDa, respectively. Circular dichroism data of the rAOx confirmed its ordered structure. Docking studies with an ab-initio protein model demonstrated the presence of a conserved FAD binding domain with an active substrate binding site. The rAOx was specific for aryl alcohols and the order of its substrate preference was 4-methoxybenzyl alcohol >3-methoxybenzyl alcohol>3, 4-dimethoxybenzyl alcohol > benzyl alcohol. A significantly high aggregation to ∼1000 nm (diameter) and catalytic efficiency (kcat/Km) of 7829.5 min-1 mM-1 for 4-methoxybenzyl alcohol was also demonstrated for rAOx. The results infer the novelty of the AOx and its potential biocatalytic application.

Decontamination with chlorhexidine gluconate reduces the incidence of ventilator associated pneumonia.

Ventilator-associated pneumonia is a major source of nosocomical infection affecting about 27 percent of all critically ill patients. In this study, an attempt was made to assess the effect of oral decontamination with 0.2% chlorhexidine gluconate solution and hydrogen peroxide solution on the incidence of ventilator-associated pneumonia and oropharyngal colonisation. Seventy patients above 18 years of age admitted in AIIMS, New Delhi were included as subjects. It was concluded that incidence of ventilator-associated pneumonia was higher in patients given oral care with hydrogen peroxide than those on 0.2% chlorhexidine, which was more effective in reducing orophyryngeal colonisation.