A Raman Microspectroscopy Study of Water and Trehalose in Spin-Dried Cells

AbstractLong-term storage of desiccated nucleated mammalian cells at ambient temperature may be accomplished in a stable glassy state, which can be achieved by removal of water from the biological sample in the presence of glass-forming agents including trehalose. The stability of the glass may be compromised due to a nonuniform distribution of residual water and trehalose within and around the desiccated cells. Thus, quantification of water and trehalose contents at the single-cell level is critical for predicting the glass formation and stability for dry storage. Using Raman microspectroscopy, we estimated the trehalose and residual water contents in the microenvironment of spin-dried cells. Individual cells with or without intracellular trehalose were embedded in a solid thin layer of extracellular trehalose after spin-drying. We found strong evidence suggesting that the residual water was bound at a 2:1 water/trehalose molar ratio in both the extracellular and intracellular milieus. Other than the water associated with trehalose, we did not find any more residual water in the spin-dried sample, intra- or extracellularly. The extracellular trehalose film exhibited characteristics of an amorphous state with a glass transition temperature of ∼22°C. The intracellular milieu also dried to levels suitable for glass formation at room temperature. These findings demonstrate a method for quantification of water and trehalose in desiccated specimens using confocal Raman microspectroscopy. This approach has broad use in desiccation studies to carefully investigate the relationship of water and trehalose content and distribution with the tolerance to drying in mammalian cells

Effect of trehalose as an additive to dimethyl sulfoxide solutions on ice formation, cellular viability, and metabolism.

Cryopreservation is the only established method for long-term preservation of cells and cellular material. This technique involves preservation of cells and cellular components in the presence of cryoprotective agents (CPAs) at liquid nitrogen temperatures (−196 °C). The organic solvent dimethyl sulfoxide (Me2SO) is one of the most commonly utilized CPAs and has been used with various levels of success depending on the type of cells. In recent years, to improve cryogenic outcomes, the non-reducing disaccharide trehalose has been used as an additive to Me2SO-based freezing solutions. Trehalose is a naturally occurring non-toxic compound found in bacteria, fungi, plants, and invertebrates which has been shown to provide cellular protection during water-limited states. The mechanism by which trehalose improves cryopreservation outcomes remains not fully understood. Raman microspectroscopy is a powerful tool to provide valuable insight into the nature of interactions among water, trehalose, and Me2SO during cryopreservation. We found that the addition of trehalose to Me2SO based CPA solutions dramatically reduces the area per ice crystals while increasing the number of ice crystals formed when cooled to −40 or −80 °C. Differences in ice-formation patterns were found to have a direct impact on cellular viability. Despite the osmotic stress caused by addition of 100 mM trehalose, improvement in cellular viability was observed. However, the substantial increase in osmotic pressure caused by trehalose concentrations above 100 mM may offset the beneficial effects of changing the morphology of the ice crystals achieved by addition of this sugar

Modulation of cellular energetics by galactose and pioglitazone.

The Warburg effect is ameliorated by culturing transformed cells in the presence of galactose instead of glucose as the primary carbon source. However, metabolic consequences that are in addition to sensitizing the cells to mitochondrial toxins may occur. As such, the screening of pharmaceutical agents against transformed cells while using galactose must be carefully evaluated. Pioglitazone is used in clinical applications to treat type-2 diabetes, but clearly has other off target effects. Human hepatocellular carcinoma cells (HepG2) were cultured in glucose or galactosecontaining medium to investigate the role of pioglitazone on cellular bioenergetics employing calorimetry and respirometry. Compared to cells cultured in 10 mM glucose, HepG2 cells cultured in the presence of 10 mM galactose showed decreased metabolic activity as measured by cellular heat flow. Interestingly, cellular heat flow increased after addition of pioglitazone for cells cultured in glucose, but not for cells cultured in galactose. Our calorimetric data indicate that a reduction in cellular capacity for glycolysis might be the mechanism responsible for the increase in sensitivity to pioglitazone, and likely mitochondrial toxins in general, for cells cultured in galactose. Furthermore, oxygen consumption rates were decreased after addition of pioglitazone to cells grown in glucose, but remained unchanged for cells grown in presence of galactose. Taken together, we demonstrate that pioglitazone induced a reduction in mitochondrial activity that was partially compensated via an increase in glycolysis in the presence of glucose

Trehalose Transporter from African Chironomid Larvae Improves Desiccation Tolerance of Chinese Hamster Ovary Cells

Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation

Continuous gas processing without bubbles using thin liquid film bioreactors containing biocomposite biocatalysts

Continuous microbial gas processing without bubbles is possible with thin liquid film, plug flow bioreactors. We have demonstrated that power input can be minimized by using a falling liquid film operating under laminar wavy flow conditions (Re \u3c200) in contact with highly concentrated living, non-growing microbes stabilized in a porous biocomposite biocatalyst. This composite materials approach to continuous gas processing can dramatically increase mass transfer rates \u3e100 fold compared to bubble aeration, decrease process volume, significantly decrease gas-liquid mass transfer energy input, decrease water use, and increase secreted product concentration. We have shown that this approach can also increase microbial specific activity for some organisms compared to microbes suspended in liquid media. Paper-based biocomposite biocatalysts provide a rough hydrophilic surface resulting in uniform ~300 μm thick falling liquid films. Paper roughness enhances gas-liquid-microbe mass transfer. This mass transfer enhancement has been simulated using a finite element (FEM) CFD model. The paper structure also functions as a separation device - the secreted products are released into the falling liquid film and continuously removed from the reactor. We are investigating biocomposite biocatalyst design and stabilization using a 0.05 m2 prototype cylindrical paper falling film bioreactor (FFBR). This approach can be used for continuous gas processing with either non-photosynthetic or photosynthetic microorganisms. Current experimental model systems we are investigating include Clostridium ljungdahlii OTA1 for absorbing CO from syn-gas, Methylomycrobium alkaliphilum 20Z for absorbing CH4 in air, and Chlamydomonas renhardtii for CO2 emissions. Critical to biocomposite biocatalyst design are generation of nanoporous coating microstructure, microbe adhesion to paper during film formation (which may include engineering the surface of the microbes), surviving osmotic shock in coating formulations, as well as desiccation tolerance to drying and prolonged dry storage. Spatially correlated Raman microspectroscopy and hyperspectral imaging techniques have been developed as a non-destructive method to monitor the distribution of residual water surrounding and within the cells. The distribution of vitrified residual water may contribute to desiccation resistance. Other types of thin liquid film reactors, such as a spinning disk bioreactor (SDBR), that enhance mass transfer by reducing liquid film thickness to \u3c100 μm with wave induced turbulent flow using centrifugal force (1000 x g) can be used in the future to further intensify continuous gas processing rates using biocomposite biocatalysts

Preclinical hematological profile studies of an ayurvedic medicine Siddha Makardhwaja after chronic administration to male sprague-dawley rats

Background: Siddha Makardhwaja (SMD) is a classical Ayurvedic formulation markedly used as a traditional medicine in the rural population for various purposes such as stimulant, tonic, and rejuvenator.Methods: The present study is conducted to evaluate the effect of conventionally prepared SMD on the hematological parameters in experimental animals, for providing scientific data base for its logical use in clinical practice. Acute toxicity tests were conducted to determine the LD50 of the drug. To find out the effect of chronic administration of SMD on hematological parameters it was administered chronically to the male Sprague-Dawley rats at a dose of 40mg/kg for 28 days.Results: In this experiment the TC, DC, various erythrocytic parameters, platelet parameters, ESR were determined. The results of the studies are given below. There is an (13.41%) increase in the number of white blood cell count of the male rat, the increase though not significant yet it was prominent (p=0.257). There is an (15.87%) increase in the absolute count of Neutrophils of the male rat, the increase though not significant yet it was prominent (p=0.371). There is an (12.29%) increase in the absolute count of Lymphocytes of the male rat, the increase though not significant yet it was prominent (p=0.388). There is a statistically significant (p=0.035) increase in the number of platelet count of the male rat (11.13% increase). There is a (2.03%) decrease in the platelet volume distribution width of the male rat, the decrease though not significant yet it was noticeable (p=0.094). There is a statistically insignificant (p=0.619) (10.0%) increase in Erythrocyte sedimentation rate in blood from the male rat.Conclusions: As SMD decreases and increases abnormally on the hematological parameters in body of treated rats, so it should not be administered chronically at a higher dose. Further studies should be done by reducing the administered dose

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Leviathan on trial: Should states be held criminally responsible?

AbstractMany political theorists, philosophers, and International Relations scholars argue that states are ‘corporate moral agents’, which can be held responsible in many of the same ways as individual moral agents. States can have debts, contractual obligations, reparative obligations, and duties. Should states also be subject to criminal responsibility and punishment? Thus far, the debate about state crime has focused on two general problems with corporate crime: whether corporate entities can have intentions (or mens rea); and whether it is possible to punish them. In this paper, I identify two problems with extending corporate criminal responsibility to the state. First, since there is no ‘international corporate law’ that regulates the internal structures of states, many states fail to meet the conditions for corporate agency (and hence for criminal responsibility). Second, since the most serious international crimes are not subject to a statute of limitations, the argument for state crime paves the way for forms of ‘historical punishment’ that few of its proponents would accept. Finally, I argue that it is unnecessary to hold states criminally responsible, and that state responsibility ought to be understood as reparative rather than punitive.Non