Electroporation enhances cell death in 3D scaffold-based MDA-MB-231 cells treated with metformin.

Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer lacks estrogen, progesterone, and HER2 receptors and hence, is therapeutically challenging. Towards this, we studied an alternate therapy by repurposing metformin (FDA-approved type-2 diabetic drug with anticancer properties) in a 3D-scaffold culture, with electrical pulses. 3D cell culture was used to simulate the tumor microenvironment more closely and MDA-MB-231, human TNBC cells, treated with both 5 mM metformin (Met) and 8 electrical pulses at 2500 V/cm, 10 µs (EP1) and 800 V/cm, 100 µs (EP2) at 1 Hz were studied in 3D and 2D. They were characterized using cell viability, reactive oxygen species (ROS), glucose uptake, and lactate production assays at 24 h. Cell viability, as low as 20 % was obtained with EP1 + 5 mM Met. They exhibited 1.65-fold lower cell viability than 2D with EP1 + 5 mM Met. ROS levels indicated a 2-fold increase in oxidative stress for EP1 + 5 mM Met, while the glucose uptake was limited to only 9 %. No significant change in the lactate production indicated glycolytic arrest and a non-conducive environment for MDA-MB-231 growth. Our results indicate that 3D cell culture, with a more realistic tumor environment that enhances cell death using metformin and electrical pulses could be a promising approach for TNBC therapeutic intervention studies.

Efficacy of metformin and electrical pulses in breast cancer MDA-MB-231 cells.

Aim: Triple-negative breast cancer (TNBC) is a very aggressive subset of breast cancer, with limited treatment options, due to the lack of three commonly targeted receptors, which merits the need for novel treatments for TNBC. Towards this need, the use of metformin (Met), the most widely used type-2 diabetes drug worldwide, was explored as a repurposed anticancer agent. Cancer being a metabolic disease, the modulation of two crucial metabolites, glucose, and reactive oxygen species (ROS), is studied in MDA-MB-231 TNBC cells, using Met in the presence of electrical pulses (EP) to enhance the drug efficacy. Methods: MDA-MB-231, human TNBC cells were treated with Met in the presence of EP, with various concentrations Met of 1 mmol/L, 2.5 mmol/L, 5 mmol/L, and 10 mmol/L. EP of 500 V/cm, 800 V/cm, and 1,000 V/cm (with a pulse width of 100 µs at 1 s intervals) were applied to TNBC and the impact of these two treatments was studied. Various assays, including cell viability, microscopic inspection, glucose, ROS, and wound healing assay, were performed to characterize the response of the cells to the combination treatment. Results: Combining 1,000 V/cm with 5 mmol/L Met yielded cell viability as low as 42.6% at 24 h. The glucose level was reduced by 5.60-fold and the ROS levels were increased by 9.56-fold compared to the control, leading to apoptotic cell death. Conclusions: The results indicate the enhanced anticancer effect of Met in the presence of electric pulses. The cell growth is inhibited by suppressing glucose levels and elevated ROS. This shows a synergistic interplay between electroporation, Met, glucose, and ROS metabolic alterations. The results show promises for combinational therapy in TNBC patients.

Adult Cerebral Malaria: Acute and Subacute Imaging Findings, Long-term Clinical Consequences.

Cerebral malaria is an important cause of mortality and neurodisability in endemic regions. We show magnetic resonance imaging (MRI) features suggestive of cytotoxic and vasogenic cerebral edema followed by microhemorrhages in 2 adult UK cases, comparing them with an Indian cohort. Long-term follow-up images correlate ongoing changes with residual functional impairment.

The effectiveness of malaria camps as part of the malaria control program in Odisha, India.

Durgama Anchalare Malaria Nirakaran (DAMaN) is a multi-component malaria intervention for hard-to-reach villages in Odisha, India. The main component, malaria camps (MCs), consists of mass screening, treatment, education, and intensified vector control. We evaluated MC effectiveness using a quasi-experimental cluster-assigned stepped-wedge study with a pretest-posttest control group in 15 villages: six immediate (Arm A), six delayed (Arm B), and three previous interventions (Arm C). The primary outcome was PCR + Plasmodium infection prevalence. The time (i.e., baseline vs. follow-up 3) x study arm interaction term shows that there were statistically significant lower odds of PCR + Plasmodium infection in Arm A (AOR = 0.36, 95% CI = 0.17, 0.74) but not Arm C as compared to Arm B at the third follow-up. The cost per person ranged between US$3-8, the cost per tested US$4-9, and the cost per treated US$82-1,614, per camp round. These results suggest that the DAMaN intervention is a promising and financially feasible approach for malaria control.

Pathogenesis of Cerebral Malaria: New Trends and Insights for Developing Adjunctive Therapies.

No specific or adjunctive therapies exist to treat cerebral malaria (CM) as of date. CM is a neuropathological manifestation of the malaria infection in humans, caused by the hemoparasitic pathogen Plasmodium falciparum. Driven through a multitude of virulence factors, varied immune responses, variations in brain swelling with regard to the age of patients, parasite biomass, and parasite-typing, the essential pathogenetic mechanisms underlying clinical CM have remained elusive. However, a recent series of studies based on molecular, immunologic, and advanced neuroradiologic and machine-learning approaches have unraveled new trends and insights to better understand and focus on the key determinants of CM in humans. This could possibly be the beginning of the design of new and effective adjunctive therapies that may not be common or applicable to the entire malarious world, but that could, rather, be specific to the variations in the determinants of CM.

The effectiveness of malaria camps as part of the malaria control program in Odisha, India.

Durgama Anchalare Malaria Nirakaran (DAMaN) is a multi-component malaria intervention for hard-to-reach villages in Odisha, India. The main component, Malaria Camps (MCs), consists of mass screening, treatment, education, and intensified vector control. We evaluated MC effectiveness using a quasi-experimental cluster-assigned stepped-wedge study with a pretest-posttest control group in 15 villages: six immediate (Arm A), six delayed (Arm B), and three previous interventions (Arm C). The primary outcome was PCR+ Plasmodium infection prevalence. Across all arms, the odds of PCR+ malaria were 54% lower at the third follow-up compared to baseline. A time (i.e., visit) x study arm interaction revealed significantly lower odds of PCR+ malaria in Arm A versus B at the third follow-up. The cost per person ranged between US$3-8, the cost per tested US$4-7, and the cost per treated US$82-1,614, per camp round. These results suggest that the DAMaN intervention is a promising, financially feasible approach for malaria control.

Advances in Basic and Translational Research as Part of the Center for the Study of Complex Malaria in India.

The Center for the Study of Complex Malaria in India (CSCMi) is one of 10 International Centers of Excellence in Malaria Research funded by the National Institutes of Health since 2010. The Center combines innovative research with capacity building and technology transfer to undertake studies with clinical and translational impact that will move malaria control in India toward the ultimate goal of malaria elimination/eradication. A key element of each research site in the four states of India (Tamil Nadu, Gujarat, Odisha, and Meghalaya) has been undertaking community- and clinic-based epidemiology projects to characterize the burden of malaria in the region. Demographic and clinical data and samples collected during these studies have been used in downstream projects on, for example, the widespread use of mosquito repellants, the population genomics of Plasmodium vivax, and the serological responses to P. vivax and Plasmodium falciparum antigens that reflect past or present exposure. A focus has been studying the pathogenesis of severe malaria caused by P. falciparum through magnetic resonance imaging of cerebral malaria patients. Here we provide a snapshot of some of the basic and applied research the CSCMi has undertaken over the past 12 years and indicate the further research and/or clinical and translational impact these studies have had.

The Impact, Emerging Needs, and New Research Questions Arising from 12 Years of the Center for the Study of Complex Malaria in India.

The Center for the Study of Complex Malaria in India (CSCMi) was launched in 2010 with the overall goal of addressing major gaps in our understanding of "complex malaria" in India through projects on the epidemiology, transmission, and pathogenesis of the disease. The Center was mandated to adopt an integrated approach to malaria research, including building capacity, developing infrastructure, and nurturing future malaria leaders while conducting relevant and impactful studies to assist India as it moves from control to elimination. Here, we will outline some of the interactions and impacts the Center has had with malaria policy and control counterparts in India, as well as describe emerging needs and new research questions that have become apparent over the past 12 years.

Transcellular blood-brain barrier disruption in malaria-induced reversible brain edema.

Brain swelling occurs in cerebral malaria (CM) and may either reverse or result in fatal outcome. It is currently unknown how brain swelling in CM reverses, as brain swelling at the acute stage is difficult to study in humans and animal models with reliable induction of reversible edema are not known. In this study, we show that reversible brain swelling in experimental murine CM can be induced reliably after single vaccination with radiation-attenuated sporozoites as proven by in vivo high-field magnetic resonance imaging. Our results provide evidence that brain swelling results from transcellular blood-brain barrier disruption (BBBD), as revealed by electron microscopy. This mechanism enables reversal of brain swelling but does not prevent persistent focal brain damage, evidenced by microhemorrhages, in areas of most severe BBBD. In adult CM patients magnetic resonance imaging demonstrate microhemorrhages in more than one third of patients with reversible edema, emphasizing similarities of the experimental model and human disease. Our data suggest that targeting transcellular BBBD may represent a promising adjunct therapeutic approach to reduce edema and may improve neurological outcome.

Evidence of Brain Alterations in Noncerebral Falciparum Malaria.

BACKGROUND: Cerebral malaria in adults is associated with brain hypoxic changes on magnetic resonance (MR) images and has a high fatality rate. Findings of neuroimaging studies suggest that brain involvement also occurs in patients with uncomplicated malaria (UM) or severe noncerebral malaria (SNCM) without coma, but such features were never rigorously characterized. METHODS: Twenty patients with UM and 21 with SNCM underwent MR imaging on admission and 44-72 hours later, as well as plasma analysis. Apparent diffusion coefficient (ADC) maps were generated, with values from 5 healthy individuals serving as controls. RESULTS: Patients with SNCM had a wide spectrum of cerebral ADC values, including both decreased and increased values compared with controls. Patients with low ADC values, indicating cytotoxic edema, showed hypoxic patterns similar to cerebral malaria despite the absence of deep coma. Conversely, high ADC values, indicative of mild vasogenic edema, were observed in both patients with SNCM and patients with UM. Brain involvement was confirmed by elevated circulating levels of S100B. Creatinine was negatively correlated with ADC in SNCM, suggesting an association between acute kidney injury and cytotoxic brain changes. CONCLUSIONS: Brain involvement is common in adults with SNCM and a subgroup of hospitalized patients with UM, which warrants closer neurological follow-up. Increased creatinine in SNCM may render the brain more susceptible to cytotoxic edema.

Enhanced Antiproliferation Potency of Electrical Pulse-Mediated Metformin and Cisplatin Combination Therapy on MDA-MB-231 Cells.

We investigated the combined potency of metformin and cisplatin on the MDA-MB-231, triple-negative breast cancer (TNBC) cells with the application of electrical pulses. There are no targeted therapies for this subset of breast cancer because of the absence of specific biomarkers. Cytotoxic chemotherapy is the mainstream mode of treatment for TNBC, and cisplatin is the most commonly used chemotherapeutic drug. While there is a good response initially, TNBC cells develop drug resistance eventually. Thus, there is a need for alternate therapies. Toward this, we studied the antiproliferation characteristics of electrical pulse-mediated combination therapy using metformin, the commonly used Type-2 diabetes drug, along with cisplatin. We used metformin, as it has various anticancer properties caused by repressing energy pathways in a cancer cell. Application of 8 pulses of 1000 V/cm, 100 µs, at 1 Hz frequency, enhanced the drug uptake leading to cell viability as low as 25.86% at 30 µM cisplatin and 5 mM metformin in a 24 h study. Also, the same studies were conducted on MCF10A, a non-cancerous human epithelial cell. It aided in comparing the result for both MDA-MB-231 and MCF10A cell lines while establishing a better understanding of the experimental outcomes. Overall, the various experimental results from colony-forming assay, reactive oxidative analysis, and the intracellular glucose metabolic assay indicate the possibility of the electrical pulses-based cisplatin and metformin drug combination as a potential alternative to TNBC treatment.

Determinants of brain swelling in pediatric and adult cerebral malaria.

Cerebral malaria (CM) affects children and adults, but brain swelling is more severe in children. To investigate features associated with brain swelling in malaria, we performed blood profiling and brain MRI in a cohort of pediatric and adult patients with CM in Rourkela, India, and compared them with an African pediatric CM cohort in Malawi. We determined that higher plasma Plasmodium falciparum histidine rich protein 2 (PfHRP2) levels and elevated var transcripts that encode for binding to endothelial protein C receptor (EPCR) were linked to CM at both sites. Machine learning models trained on the African pediatric cohort could classify brain swelling in Indian children CM cases but had weaker performance for adult classification, due to overall lower parasite var transcript levels in this age group and more severe thrombocytopenia in Rourkela adults. Subgrouping of patients with CM revealed higher parasite biomass linked to severe thrombocytopenia and higher Group A-EPCR var transcripts in mild thrombocytopenia. Overall, these findings provide evidence that higher parasite biomass and a subset of Group A-EPCR binding variants are common features in children and adult CM cases, despite age differences in brain swelling.

The effectiveness of malaria camps as part of the Durgama Anchalare Malaria Nirakaran (DAMaN) program in Odisha, India: study protocol for a cluster-assigned quasi-experimental study.

The Indian state of Odisha has a longstanding battle with forest malaria. Many remote and rural villages have poor access to health care, a problem that is exacerbated during the rainy season when malaria transmission is at its peak. Approximately 62% of the rural population consists of tribal groups who are among the communities most negatively impacted by malaria. To address the persistently high rates of malaria in these remote regions, the Odisha State Malaria Control Program introduced 'malaria camps' in 2017 where teams of health workers visit villages to educate the population, enhance vector control methods, and perform village-wide screening and treatment. Malaria rates declined statewide, particularly in forested areas, following the introduction of the malaria camps, but the impact of the intervention is yet to be externally evaluated. This study protocol describes a cluster-assigned quasi-experimental stepped-wedge study with a pretest-posttest control group design that evaluates if malaria camps reduce the prevalence of malaria, compared to control villages which receive the usual malaria control interventions (e.g. IRS, ITNs), as detected by PCR.

Plasmodium falciparum erythrocyte membrane protein 1 variants induce cell swelling and disrupt the blood-brain barrier in cerebral malaria.

Cerebral malaria (CM) is caused by the binding of Plasmodium falciparum-infected erythrocytes (IEs) to the brain microvasculature, leading to inflammation, vessel occlusion, and cerebral swelling. We have previously linked dual intercellular adhesion molecule-1 (ICAM-1)- and endothelial protein C receptor (EPCR)-binding P. falciparum parasites to these symptoms, but the mechanism driving the pathogenesis has not been identified. Here, we used a 3D spheroid model of the blood-brain barrier (BBB) to determine unexpected new features of IEs expressing the dual-receptor binding PfEMP1 parasite proteins. Analysis of multiple parasite lines shows that IEs are taken up by brain endothelial cells in an ICAM-1-dependent manner, resulting in breakdown of the BBB and swelling of the endothelial cells. Via ex vivo analysis of postmortem tissue samples from CM patients, we confirmed the presence of parasites within brain endothelial cells. Importantly, this discovery points to parasite ingress into the brain endothelium as a contributing factor to the pathology of human CM.

Repaglinide and Metformin-Loaded Amberlite Resin-Based Floating Microspheres for the Effective Management of Type 2 Diabetes.

BACKGROUND: Low bioavailability of anti-diabetic drugs results in the partial absorption of the drug as they are mainly absorbed from the stomach and the lower part of the GIT. Drug bioavailability of anti-diabetic drugs can be significantly increased by prolonging gastric retention time through gastro-retentive dosage form such as floating microspheres. OBJECTIVE: The study was aimed to develop and characterize resin based floating microspheres of Repaglinide and Metformin for superior and prolonged maintenance of normoglycaemia in type-2 diabetes mellitus. METHODS: Repaglinide and metformin were complexed with amberlite resin; later resin complexed drug was encapsulated in Ethylcellulose floating microspheres. Floating microspheres were characterized for morphology, particle size, IR spectroscopy, DSC, in vitro release and buoyancy studies. Further, floating microspheres were tested for in vivo blood glucose reduction potential in Streptozocin- induced diabetic mice. RESULTS: Floating microspheres had a spherical shape and slightly rough surface with the entrapment efficiency in a range of 49-78% for metformin and 52-73% for repaglinide. DSC studies revealed that no chemical interaction took place between polymer and drugs. Floating microspheres showed good buoyancy behavior (P<0.05) and prolonged drug release as compared to plain drug (P<0.05). The blood glucose lowering effect of floating microspheres on Streptozocin induced diabetic rats was significantly greater (P<0.05) and prolonged (˃12h) and normoglycaemia was maintained for 6hr. CONCLUSION: Floating microspheres containing drug resin complex were able to prolong drug release in an efficient way for a sustained period of time; as a result, profound therapeutic response was obtained.

Brain Magnetic Resonance Imaging Reveals Different Courses of Disease in Pediatric and Adult Cerebral Malaria.

BACKGROUND: Cerebral malaria is a common presentation of severe Plasmodium falciparum infection and remains an important cause of death in the tropics. Key aspects of its pathogenesis are still incompletely understood, but severe brain swelling identified by magnetic resonance imaging (MRI) was associated with a fatal outcome in African children. In contrast, neuroimaging investigations failed to identify cerebral features associated with fatality in Asian adults. METHODS: Quantitative MRI with brain volume assessment and apparent diffusion coefficient (ADC) histogram analyses were performed for the first time in 65 patients with cerebral malaria to compare disease signatures between children and adults from the same cohort, as well as between fatal and nonfatal cases. RESULTS: We found an age-dependent decrease in brain swelling during acute cerebral malaria, and brain volumes did not differ between fatal and nonfatal cases across both age groups. In nonfatal disease, reversible, hypoxia-induced cytotoxic edema occurred predominantly in the white matter in children, and in the basal ganglia in adults. In fatal cases, quantitative ADC histogram analyses also demonstrated different end-stage patterns between adults and children: Severe hypoxia, evidenced by global ADC decrease and elevated plasma levels of lipocalin-2 and microRNA-150, was associated with a fatal outcome in adults. In fatal pediatric disease, our results corroborate an increase in brain volume, leading to augmented cerebral pressure, brainstem herniation, and death. CONCLUSIONS: Our findings suggest distinct pathogenic patterns in pediatric and adult cerebral malaria with a stronger cytotoxic component in adults, supporting the development of age-specific adjunct therapies.

Polymer/Graphene oxide nanocomposite thin film for NO2 sensor: An in situ investigation of electronic, morphological, structural, and spectroscopic properties.

The higher operating temperature of metal oxide and air instability of organic based NO2 sensor causes extremely urgent for development of a reliable low cost sensor to detect NO2 at room temperature. Therefore, we present a fabrication of large area Polymer/GO nano hybrid thin film for polymer thin film transistors (PTFTs) based NO2 sensors assisted via facile method named 'spreading-solidifying (SS) method', grown over air/liquid interface and successive investigation of effect of NO2 on film via several characterizations. The PTFTs sensor has demonstrated swift and high response towards low concentration of NO2 gas with air stability and provided real time non-invasive type NO2 sensor. Herein, we are reporting the nanohybrid PBTTT/GO composite based PTFT sensor with good repeatability and sensor response for low concentration NO2. The thin film grown via SS technique has reported very good adsorption/desorption of target analyte having response/recovery time of 75 s/523 s for 10 ppm concentration of NO2 gas. It has been observed that % change in drain current (sensor response) saturated with increasing concentration of NO2. The transient analysis demonstrates the fast sensor response and recovery time. Furthermore, in order to understand the insight of high performance of sensor, effect of NO2 on nanohybrid film and sensing mechanism, an in situ investigations was conducted via multiple technique viz. spectral, electronic, structural, and morphological characterization. Finally, the performance of sensor and the site of adsorption of NO2 at polymer chains were argued using schematic diagram. This work shows the simple fabrication process for mass production, low cost and room temperature operated gas sensors for monitoring the real-time environment conditions and gives an insight about the sensing mechanism adsorption site of NO2.

Malaria inflammation by xanthine oxidase-produced reactive oxygen species.

Malaria is a highly inflammatory disease caused by Plasmodium infection of host erythrocytes. However, the parasite does not induce inflammatory cytokine responses in macrophages in vitro and the source of inflammation in patients remains unclear. Here, we identify oxidative stress, which is common in malaria, as an effective trigger of the inflammatory activation of macrophages. We observed that extracellular reactive oxygen species (ROS) produced by xanthine oxidase (XO), an enzyme upregulated during malaria, induce a strong inflammatory cytokine response in primary human monocyte-derived macrophages. In malaria patients, elevated plasma XO activity correlates with high levels of inflammatory cytokines and with the development of cerebral malaria. We found that incubation of macrophages with plasma from these patients can induce a XO-dependent inflammatory cytokine response, identifying a host factor as a trigger for inflammation in malaria. XO-produced ROS also increase the synthesis of pro-IL-1ß, while the parasite activates caspase-1, providing the two necessary signals for the activation of the NLRP3 inflammasome. We propose that XO-produced ROS are a key factor for the trigger of inflammation during malaria.

An efficient, mild and metal free l-proline catalyzed construction of fused pyrimidines under microwave conditions in water.

One-pot condensation of 4-hydroxy coumarins, aldehydes and urea/thiourea to build C-C and C-N bonds is described. Fused pyrimidines have been synthesized under mild reaction conditions using l-proline. The protocol has been performed rapidly and efficiently in water under metal free conditions. Heterocyclic derivatives have been synthesized using the present methodology and avoid the use of hazardous solvents over conventional organic solvents. A proposed mechanism could be established for three component reactions. The present study reveals the first case in which l-proline has been explored as a homogeneous catalyst in the synthesis of fused pyrimidines in water under microwave irradiation. This synthesis involves simple workup and acceptable efficiency. The most notable feature of this protocol is the ability of the catalyst to influence asymmetric induction in the reaction.