Differential dose-response effect of cyclosporine A in regulating apoptosis and autophagy markers in MCF-7 cells.

Cyclosporine A (CsA) is an immunosuppressant primarily used at a higher dosage in transplant medicine and autoimmune diseases with a higher success rate. At lower doses, CsA exhibits immunomodulatory properties. CsA has also been reported to inhibit breast cancer cell growth by downregulating the expression of pyruvate kinase. However, differential dose-response effects of CsA in cell growth, colonization, apoptosis, and autophagy remain largely unidentified in breast cancer cells. Herein, we showed the cell growth-inhibiting effects of CsA by preventing cell colonization and enhancing DNA damage and apoptotic index at a relatively lower concentration of 2 µM in MCF-7 breast cancer cells. However, at a higher concentration of 20 µM, CsA leads to differential expression of autophagy-related genes ATG1, ATG8, and ATG9 and apoptosis-associated markers, such as Bcl-2, Bcl-XL, Bad, and Bax, indicating a dose-response effect on differential cell death mechanisms in MCF-7 cells. This was confirmed in the protein-protein interaction network of COX-2 (PTGS2), a prime target of CsA, which had close interactions with Bcl-2, p53, EGFR, and STAT3. Furthermore, we investigated the combined effect of CsA with SHP2/PI3K-AKT inhibitors showing significant MCF-7 cell growth reduction, suggesting its potential to use as an adjuvant during breast cancer therapy.

Plant Flavonoids on Oxidative Stress-Mediated Kidney Inflammation.

The kidney is susceptible to reactive oxygen species-mediated cellular injury resulting in glomerulosclerosis, tubulointerstitial fibrosis, tubular cell apoptosis, and senescence, leading to renal failure, and is a significant cause of death worldwide. Oxidative stress-mediated inflammation is a key player in the pathophysiology of various renal injuries and diseases. Recently, flavonoids' role in alleviating kidney diseases has been reported with an inverse correlation between dietary flavonoids and kidney injuries. Flavonoids are plant polyphenols possessing several health benefits and are distributed in plants from roots to leaves, flowers, and fruits. Dietary flavonoids have potent antioxidant and free-radical scavenging properties and play essential roles in disease prevention. Flavonoids exert a nephroprotective effect by improving antioxidant status, ameliorating excessive reactive oxygen species (ROS) levels, and reducing oxidative stress, by acting as Nrf2 antioxidant response mediators. Moreover, flavonoids play essential roles in reducing chemical toxicity. Several studies have demonstrated the effects of flavonoids in reducing oxidative stress, preventing DNA damage, reducing inflammatory cytokines, and inhibiting apoptosis-mediated cell death, thereby preventing or improving kidney injuries/diseases. This review covers the recent nephroprotective effects of flavonoids against oxidative stress-mediated inflammation in the kidney and their clinical advancements in renal therapy.

Adenosine Triphosphate Protects from Elevated Extracellular Calcium-Induced Damage in Human Proximal Kidney Cells: Using Deep Learning to Predict Cytotoxicity.

BACKGROUND/AIMS: In kidney, extracellular [Ca2+] can modulate intracellular [Ca2+] to control key cellular processes. Hence, extracellular [Ca2+] is normally maintained within narrow range. We tested effect of extracellular ATP on viability of human proximal (HK-2) cells at high calcium. Modulation of intracellular calcium was assessed by imaging cytosolic [Ca2+], and expression of calcium-binding proteins (CaBPs). We present an artificial intelligence enabled deep learning model for prediction of injury and protection against extracellular [Ca2+] in HK-2 cells. METHODS: HK-2 cells were cultured in calcium-free DMEM supplemented with CaCl2. Morphological changes were detected using light microscopy. Cell viability was determined using MTT Assay. Intracellular [Ca2+] was detected using fluorescence microscopy. For easy detection of HK-2 cells injury, we performed light microscopy image classification based on Convolutional Neural Network. Expression of CaBPs, p21, and Mcl-1 was measured using real-time PCR. RESULTS: We show decreased viability of HK-2 cells cultured in elevated calcium levels, which was prevented by adenosine triphosphate (ATP). Exposure of cells to elevated extracellular [Ca2+] correlated with increasing fluorescence of intracellular calcium indicator, which was attenuated in presence of ATP. Since features cannot be detected easily by human eyes, we propose a customized deep learning-based CNN model for classification of HK-2 cells injury by extracellular calcium with high accuracy of 98%. Our data demonstrated significant increase in mRNA levels of calmodulin, S100A8, S100A14 and CaBP28k, with elevated extracellular [Ca2+]. Expression of these genes was enhanced with ATP. CONCLUSION: The results suggest that ATP protects human proximal (HK-2) cells against elevated extracellular calcium levels. We present a CNN model as user friendly tool to study calcium dependent injury in (HK-2) cells. Finally, we show that ATP-mediated protection is correlated with enhanced expression of calcium-binding proteins.

Current methodologies to refine bioavailability, delivery, and therapeutic efficacy of plant flavonoids in cancer treatment.

Over the last two decades, several advancements have been made to improve the therapeutic efficacy of plant flavonoids, especially in cancer treatment. Factors such as low bioavailability, poor flavonoid stability and solubility, ineffective targeted delivery, and chemo-resistance hinder the application of flavonoids in anti-cancer therapy. Many anti-cancer compounds failed in the clinical trials because of unexpected altered clearance of flavonoids, poor absorption after administration, low efficacy, and/or adverse effects. Hence, the current research strategies are focused on improving the therapeutic efficacy of plant flavonoids, especially by enhancing their bioavailability through combination therapy, engineering gut microbiota, regulating flavonoids interaction with adenosine triphosphate binding cassette efflux transporters, and efficient delivery using nanocrystal and encapsulation technologies. This review aims to discuss different methodologies with examples from reported dietary flavonoids that showed an enhanced anti-cancer efficacy in both in vitro and in vivo models. Further, the review discusses the recent progress in biochemical modifications of flavonoids to improve bioavailability, solubility, and therapeutic efficacy.

Modulation of calcium-binding proteins expression and cisplatin chemosensitivity by calcium chelation in human breast cancer MCF-7 cells.

Cisplatin (CDDP) is currently one of the most effective FDA-approved treatments for breast cancer. Previous studies have shown that CDDP-induced cell death in human breast cancer (MCF-7) cells is associated with disruption of calcium homeostasis. However, whether the sensitivity of breast cancer cells to cisplatin is associated with dysregulation of the expression of calcium-binding proteins (CaBPs) remains unknown. In this study, we evaluated the effect of the intracellular calcium chelator (BAPTA-AM) on viability of MCF-7 cells in the presence of toxic and sub-toxic doses of cisplatin. Furthermore, this study assessed the expression of CaBPs, calmodulin, S100A8, and S100A14 in MCF-7 cells treated with cisplatin. Cell viability was determined using MTT-based in vitro toxicity assay. Intracellular calcium imaging was done using Fluo-4 AM, a cell-permeant fluorescent calcium indicator. Expression of CaBPs was tested using real-time quantitative PCR. Exposure of cells to increasing amounts of CDDP correlated with increasing fluorescence of the intracellular calcium indicator, Fluo-4 AM. Conversely, treating cells with cisplatin significantly decreased mRNA levels of calmodulin, S100A8, and S100A14. Treatment of the cells with calcium chelator, BAPTA-AM, significantly enhanced the cytotoxic effects of sub-toxic dose of cisplatin. Our results indicated a statistically significant negative correlation between calmodulin, S100A8, and S100A14 expression and sensitivity of breast cancer cells to a sub-toxic dose of cisplatin. We propose that modulating the activity of calcium-binding proteins, calmodulin, S100A8, and S100A14, could be used to increase cisplatin efficacy, lowering its treatment dosage while maintaining its chemotherapeutic value.

Tools to study and target the Siglec-sialic acid axis in cancer.

Siglecs are widely expressed on leucocytes and bind to ubiquitously presented glycans containing sialic acids (sialoglycans). Most Siglecs carry an immunoreceptor tyrosine-based inhibition motif (ITIM) and elicit an inhibitory intracellular signal upon ligand binding. A few Siglec receptors can, however, recruit immunoreceptor tyrosine-based activation motif (ITAM)-containing factors, which activate cells. The role of hypersialylation (the enhanced expression of sialoglycans) has recently been explored in cancer progression. Mechanistic studies have shown that hypersialylation on cancer cells can engage inhibitory Siglecs on the surface of immune cells and induce immunosuppression. These recent studies strongly suggest that the Siglec-sialic acid axis can act as a potential target for cancer immunotherapy. Moreover, the use of new tools and techniques is facilitating these studies. In this review, we summarise techniques used to study Siglecs, including different mouse models, monoclonal antibodies, Siglec fusion proteins, and sialoglycan arrays. Furthermore, we discuss the recent major developments in the study of Siglecs in cancer immunosuppression, tools, and techniques used in targeting the Siglec-sialic acid axis and the possibility of clinical intervention.

Natural compound catechol induces DNA damage, apoptosis, and G1 cell cycle arrest in breast cancer cells.

Targeting cell cycle and inducing DNA damage by activating cell death pathways are considered as effective therapeutic strategy for combating breast cancer progression. Many of the naturally known small molecules target these signaling pathways and are effective against resistant and/or aggressive types of breast cancers. Here, we investigated the effect of catechol, a naturally occurring plant compound, for its specificity and chemotherapeutic efficacies in breast cancer (MCF-7 and MDA-MB-231) cells. Catechol treatment showed concentration-dependent cytotoxicity and antiproliferative growth in both MCF-7 and MDA-MB-231 cells while sparing minimal effects on noncancerous (F-180 and HK2) cells. Catechol modulated differential DNA damage effects by activating ATM/ATR pathways and showed enhanced γ-H2AX expression, as an indicator for DNA double-stranded breaks. MCF-7 cells showed G1 cell cycle arrest by regulating p21-mediated cyclin E/Cdk2 inhibition. Furthermore, activation of p53 triggered a caspase-mediated cell death mechanism by inhibiting regulatory proteins such as DNMT1, p-BRCA1, MCL-1, and PDCD6 with an increased Bax/Bcl-2 ratio. Overall, our results showed that catechol possesses favorable safety profile for noncancerous cells while specifically targeting multiple signaling cascades to inhibit proliferation in breast cancer cells.

Siglecs in Brain Function and Neurological Disorders.

Siglecs (Sialic acid-binding immunoglobulin-type lectins) are a I-type lectin that typically binds sialic acid. Siglecs are predominantly expressed in immune cells and generate activating or inhibitory signals. They are also shown to be expressed on the surface of cells in the nervous system and have been shown to play central roles in neuroinflammation. There has been a plethora of reviews outlining the studies pertaining to Siglecs in immune cells. However, this review aims to compile the articles on the role of Siglecs in brain function and neurological disorders. In humans, the most abundant Siglecs are CD33 (Siglec-3), Siglec-4 (myelin-associated glycoprotein/MAG), and Siglec-11, Whereas in mice the most abundant are Siglec-1 (sialoadhesin), Siglec-2 (CD22), Siglec-E, Siglec-F, and Siglec-H. This review is divided into three parts. Firstly, we discuss the general biological aspects of Siglecs that are expressed in nervous tissue. Secondly, we discuss about the role of Siglecs in brain function and molecular mechanism for their function. Finally, we collate the available information on Siglecs and neurological disorders. It is intriguing to study this family of proteins in neurological disorders because they carry immunoinhibitory and immunoactivating motifs that can be vital in neuroinflammation.

Role of flavonoids in thrombotic, cardiovascular, and inflammatory diseases.

The failure of mechanisms of natural anti-coagulation either due to genetic impairment or due to severe external injuries may result in a condition called thrombosis. This is believed to be the primary cause for a variety of life-threatening conditions such as: heart attack, stroke, pulmonary embolism, thrombophlebitis, and deep venous thrombosis (DVT). The growing number of these incidents requires an alternative anti-coagulant or anti-thrombotic agent that has minimal side effects and improved efficiency. For decades, plant polyphenols, especially flavonoids, were known for their vital role in preventing various diseases such as cancer. Mitigating excessive oxidative stress caused by reactive oxygen species (ROS) with anti-oxidant-rich flavonoids may reduce the risk of hyper-activation of platelets, cardiovascular diseases (CVD), pain, and thrombosis. Furthermore, flavonoids may mitigate endothelial dysfunction (ED), which generally correlates to the development of coronary artery and vascular diseases. Flavonoids also reduce the risk of atherosclerosis and atherothrombotic disease by inhibiting excessive tissue factor (TF) availability in the endothelium. Although the role of flavonoids in CVD is widely discussed, to the best of our knowledge, their role as anti-thrombotic lead has not been discussed. This review aims to focus on the biological uses of dietary flavonoids and their role in the treatment of various coagulation disorders, and may provide some potential lead to the drug discovery process in this area.

Mitochondrial DNA, a Powerful Tool to Decipher Ancient Human Civilization from Domestication to Music, and to Uncover Historical Murder Cases.

Mitochondria are unique organelles carrying their own genetic material, independent from that in the nucleus. This review will discuss the nature of mitochondrial DNA (mtDNA) and its levels in the cell, which are the key elements to consider when trying to achieve molecular identification in ancient and degraded samples. mtDNA sequence analysis has been appropriately validated and is a consistent molecular target for the examination of biological evidence encountered in forensic cases-and profiling, in certain conditions-especially for burnt bodies and degraded samples of all types. Exceptional cases and samples will be discussed in this review, such as mtDNA from leather in Beethoven's grand piano, mtDNA in mummies, and solving famous historical criminal cases. In addition, this review will be discussing the use of ancient mtDNA to understand past human diet, to trace historical civilizations and ancient trade routes, and to uncover geographical domestication origins and lineage relationships. In each topic, we will present the power of mtDNA and how, in many cases, no nuclear DNA was left, leaving mitochondrial DNA analysis as a powerful alternative. Exploring this powerful tool further will be extremely useful to modern science and researchers, due to its capabilities in providing us with previously unattainable knowledge.

Mitochondrial DNA, restoring Beethovens music.

Great ancient composers have endured many obstacles and constraints which are very difficult to understand unless we perform the restoration process of ancient music. Species identification in leather used during manufacturing is the key step to start such a restoration process in order to produce a facsimile of a museum piano. Our study reveals the species identification in the leather covering the hammer head in a piano created by Erard in 1802. This is the last existing piano similar to the piano that Beethoven used with its leather preserved in its original state. The leather sample was not present in a homogeneous piece, yet combined with glue. Using a DNA extraction method that avoids PCR inhibitors; we discovered that sheep and cattle are the origin of the combination. To identify the species in the leather, we focused on the amounts of mitochondrial DNA in both leather and glue and results have led us to the conclusion that the leather used to cover the hammer head in this piano was made of cattle hide.

Detection of DNA sequences refractory to PCR amplification using a biophysical SERRS assay (Surface Enhanced Resonant Raman Spectroscopy).

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction - based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.

Enzyme-free detection and quantification of double-stranded nucleic acids.

We have developed a fully enzyme-free SERRS hybridization assay for specific detection of double-stranded DNA sequences. Although all DNA detection methods ranging from PCR to high-throughput sequencing rely on enzymes, this method is unique for being totally non-enzymatic. The efficiency of enzymatic processes is affected by alterations, modifications, and/or quality of DNA. For instance, a limitation of most DNA polymerases is their inability to process DNA damaged by blocking lesions. As a result, enzymatic amplification and sequencing of degraded DNA often fail. In this study we succeeded in detecting and quantifying, within a mixture, relative amounts of closely related double-stranded DNA sequences from Rupicapra rupicapra (chamois) and Capra hircus (goat). The non-enzymatic SERRS assay presented here is the corner stone of a promising approach to overcome the failure of DNA polymerase when DNA is too degraded or when the concentration of polymerase inhibitors is too high. It is the first time double-stranded DNA has been detected with a truly non-enzymatic SERRS-based method. This non-enzymatic, inexpensive, rapid assay is therefore a breakthrough in nucleic acid detection.

A novel SERRS sandwich-hybridization assay to detect specific DNA target.

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.