Analysis of daily COVID-19 death bulletin data during the first two waves of the COVID-19 pandemic in Thiruvananthapuram district, Kerala, India.

Context: Coronavirus disease 2019 (COVID-19) mortality trends can help discern the pattern of outbreak evolution and systemic responses. Aim: This study aimed to explore patterns of COVID-19 deaths in Thiruvananthapuram district from 31 March 2020 to 31 December 2021. Setting and Design: Secondary data analysis of COVID-19 deaths in Thiruvananthapuram district was performed. Materials and Methods: Mortality data were obtained from the district COVID-19 control room, and deaths in the first and second waves of COVID-19 were compared. Statistical Analysis: We summarised data as proportions and medians with the inter-quartile range (IQR) and performed Chi-square tests to make comparisons wherever applicable. Results: As on 31 December 2021, 4587 COVID-19 deaths were reported in Thiruvananthapuram district, with a case fatality rate of 0.91%. We observed high mortality among older persons (66.7%) and men (56.6%). The leading cause of death was bronchopneumonia (60.6%). The majority (88.5%) had co-morbidities, commonly diabetes mellitus (54.9%). The median interval from diagnosis to hospitalisation was 4 days (IQR 2-7), and that from hospitalisation to death was 2 days (IQR 0-6). The deaths reported during the second wave were four times higher than those of the first wave with a higher proportion of deaths in the absence of co-morbidities (p < 0.001). The majority of the deceased were unvaccinated. Ecological analysis with vaccine coverage data indicated 5.4 times higher mortality among unvaccinated than those who received two vaccine doses. Conclusions: The presence of co-morbidities, an unvaccinated status, and delay in hospitalisation were important reasons for COVID-19 deaths. Primary level health providers can potentially help sustaining vaccination, expeditious referral, and monitoring of COVID-19 patients.

Deciphering CaMKII Multimerization Using Fluorescence Correlation Spectroscopy and Homo-FRET Analysis.

While kinases are typically composed of one or two subunits, calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is composed of 8-14 subunits arranged as pairs around a central core. It is not clear if the CaMKII holoenzyme functions as an assembly of independent subunits, as catalytic pairs, or as a single unit. One strategy to address this question is to genetically engineer monomeric and dimeric CaMKII and evaluate how their activity compares to the wild-type (WT) holoenzyme. Here a technique that combines fluorescence correlation spectroscopy and homo-FRET analysis was used to characterize assembly mutants of Venus-tagged CaMKIIα to identify a dimeric CaMKII. Spectroscopy was then used to compare how holoenzyme structure and function changes in response to activation with CaM in the dimeric mutant, WT-holoenzyme, and a monomeric CaMKII oligomerization-domain deletion mutant control. CaM triggered an increase in hydrodynamic volume in both WT and dimeric CaMKII without altering subunit stoichiometry or the net homo-FRET between Venus-tagged catalytic domains. Biochemical analysis revealed that the dimeric mutant also functioned like WT holoenzyme in terms of its kinase activity with an exogenous substrate, and for endogenous T286 autophosphorylation. We conclude that the fundamental functional units of CaMKII holoenzyme are paired catalytic-domains.

A comparative cost analysis of polytrauma and neurosurgery Intensive Care Units at an apex trauma care facility in India.

CONTEXT: Although Intensive Care Units (ICUs) only account for 10% of the hospital beds, they consume nearly 22% of the hospital resources. Few definitive costing studies have been conducted in Indian settings that would help determine appropriate resource allocation. AIM: The aim of this study was to evaluate and compare the cost of intensive care delivery between multispecialty and neurosurgery ICUs at an apex trauma care facility in India. MATERIALS AND METHODS: The study was conducted in a polytrauma and neurosurgery ICU at a 203-bedded Level IV trauma care facility in New Delhi, India, from May 1, 2012 to June 30, 2012. The study was cross-sectional, retrospective, and record-based. Traditional costing was used to arrive at the cost for both direct and indirect cost estimates. The cost centers included in the study were building cost, equipment cost, human resources, materials and supplies, clinical and nonclinical support services, engineering maintenance cost, and biomedical waste management. STATISTICAL ANALYSIS: Statistical analysis was performed by Fisher's two tailed t-test. RESULTS: Total cost/bed/day for the multispecialty ICU was Rs. 14,976.9/- and for the neurosurgery ICU, it was Rs. 14,306.7/-, workforce constituting nearly half of the expenditure in both ICUs. The cost center wise and overall difference in the cost among the ICUs were statistically significant. CONCLUSIONS: Quantification of expenditure in running an ICU in a trauma center would assist health-care decision makers in better allocation of resources. Although multispecialty ICUs are more cost-effective, other factors will also play a role in defining the kind of ICU that needs to be designed.

Covert Changes in CaMKII Holoenzyme Structure Identified for Activation and Subsequent Interactions.

Between 8 to 14 calcium-calmodulin (Ca(2+)/CaM) dependent protein kinase-II (CaMKII) subunits form a complex that modulates synaptic activity. In living cells, the autoinhibited holoenzyme is organized as catalytic-domain pairs distributed around a central oligomerization-domain core. The functional significance of catalytic-domain pairing is not known. In a provocative model, catalytic-domain pairing was hypothesized to prevent ATP access to catalytic sites. If correct, kinase-activity would require catalytic-domain pair separation. Simultaneous homo-FRET and fluorescence correlation spectroscopy was used to detect structural changes correlated with kinase activation under physiological conditions. Saturating Ca(2+)/CaM triggered Threonine-286 autophosphorylation and a large increase in CaMKII holoenzyme hydrodynamic volume without any appreciable change in catalytic-domain pair proximity or subunit stoichiometry. An alternative hypothesis is that two appropriately positioned Threonine-286 interaction-sites (T-sites), each located on the catalytic-domain of a pair, are required for holoenzyme interactions with target proteins. Addition of a T-site ligand, in the presence of Ca(2+)/CaM, elicited a large decrease in catalytic-domain homo-FRET, which was blocked by mutating the T-site (I205K). Apparently catalytic-domain pairing is altered to allow T-site interactions.

Fluorescence polarization and fluctuation analysis monitors subunit proximity, stoichiometry, and protein complex hydrodynamics.

Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1-10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9 ± 1.2 subunit, but values ranged from 10-14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2 ± 1.3 subunits in the holoenzyme with values ranging from 9-12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved.

Fluorescence lifetime imaging microscopy of intracellular glucose dynamics.

BACKGROUND: One of the major hurdles in studying diabetes pathophysiology is the lack of adequate methodology that allows for direct and real-time determination of glucose transport and metabolism in cells and tissues. In this article, we present a new methodology that adopts frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) to visualize and quantify the dynamics of intracellular glucose within living cells using a biosensor protein based on fluorescence resonance energy transfer (FRET). METHOD: The biosensor protein was developed by fusing a FRET pair, an AcGFP1 donor and a mCherry acceptor to N- and C- termini of a mutant glucose-binding protein (GBP), respectively. The probe was expressed and biosynthesized inside the cells, offering continuous monitoring of glucose dynamics in real time through fluorescence lifetime imaging microscopy (FLIM) measurement. RESULTS: We transfected the deoxyribonucleic acid of the AcGFP1-GBP-mCherry sensor into murine myoblast cells, C2C12, and continuously monitored the changes in intracellular glucose concentrations in response to the variation in extracellular glucose, from which we determined glucose uptake and clearance rates. The distribution of intracellular glucose concentration was also characterized. We detected a high glucose concentration in a region close to the cell membrane and a low glucose concentration in a region close to the nucleus. The monoexponential decay of AcGFP1 was distinguished using FD-FLIM. CONCLUSIONS: This work enables continuous glucose monitoring (CGM) within living cells using FD-FLIM and a biosensor protein. The sensor protein developed offers a new means for quantitatively analyzing glucose homeostasis at the cellular level. Data accumulated from these studies will help increase our understanding of the pathology of diabetes.

Visualization of human immunodeficiency virus protease inhibition using a novel Förster resonance energy transfer molecular probe.

The in vivo high-throughput screening (HTS) of human immunodeficiency virus (HIV) protease inhibitors is a significant challenge because of the lack of reliable assays that allow the visualization of HIV targets within living cells. In this study, we developed a new molecular probe that utilizes the principles of Förster resonance energy transfer (FRET) to visualize HIV-1 protease inhibition within living cells. The probe is constructed by linking two fluorescent proteins: AcGFP1 (a mutant green fluorescent protein) and mCherry (a red fluorescent protein) with an HIV-1 protease cleavable p2/p7 peptide. The cleavage of the linker peptide by HIV-1 protease leads to separation of AcGFP1 from mCherry, quenching FRET between AcGFP1 and mCherry. Conversely, the addition of a protease inhibitor prevents the cleavage of the linker peptide by the protease, allowing FRET from AcGFP1 to mCherry. Thus, HIV-1 protease inhibition can be determined by measuring the FRET signal's change generated from the probe. Both in vitro and in vivo studies demonstrated the feasibility of applying the probe for quantitative analyses of HIV-1 protease inhibition. By cotransfecting HIV-1 protease and the probe expression plasmids into 293T cells, we showed that the inhibition of HIV-1 protease by inhibitors can be visualized or quantitatively determined within living cells through ratiometric FRET microscopy imaging measurement. It is expected that this new probe will allow high-content screening (HCS) of new anti-HIV drugs.

Construction of a panel of glucose indicator proteins for continuous glucose monitoring.

The development of implantable glucose sensors for use in diabetes treatment has been pursued for decades. However, enzyme-based glucose sensors often fail in vivo. In our previous work, we engineered a novel glucose indicator protein (GIP) that can sense glucose without relying on any enzymes and cofactors. Nevertheless, this GIP is unsuitable for blood glucose monitoring due to its low dissociation constant. Here, we report a novel approach to creating a new GIP that can be used to monitor blood glucose level. By disrupting pi-pi stacking around GIP's glucose binding site through site-directed mutagenesis, we showed that GIP's dissociation constant can be manipulated from 0.026 mM to 7.86 mM. This approach yielded four GIP mutants. We showed that one of the mutants can be used to detect glucose from 0 to 32 mM, while another mutant can be employed to visualize intracellular glucose (0-200 µM) within living cells through FRET imaging microscopy measurement.

A glucose sensor protein for continuous glucose monitoring.

In vivo continuous glucose monitoring has posed a significant challenge to glucose sensor development due to the lack of reliable techniques that are non- or at least minimally-invasive. In this proof-of-concept study, we demonstrated the development of a new glucose sensor protein, AcGFP1-GBPcys-mCherry, and an optical sensor assembly, capable of generating quantifiable FRET (fluorescence resonance energy transfer) signals for glucose monitoring. Our experimental data showed that the engineered glucose sensor protein can generate measurable FRET signals in response to glucose concentrations varying from 25 to 800 µM. The sensor developed based on this protein had a shelf-life of up to 3 weeks. The sensor response was devoid of interference from compounds like galactose, fructose, lactose, mannose, and mannitol when tested at physiologically significant concentrations of these compounds. This new glucose sensor protein can potentially be used to develop implantable glucose sensors for continuous glucose monitoring.

Tailored carbon nanotubes for tissue engineering applications.

A decade of aggressive researches on carbon nanotubes (CNTs) has paved way for extending these unique nanomaterials into a wide range of applications. In the relatively new arena of nanobiotechnology, a vast majority of applications are based on CNTs, ranging from miniaturized biosensors to organ regeneration. Nevertheless, the complexity of biological systems poses a significant challenge in developing CNT-based tissue engineering applications. This review focuses on the recent developments of CNT-based tissue engineering, where the interaction between living cells/tissues and the nanotubes have been transformed into a variety of novel techniques. This integration has already resulted in a revaluation of tissue engineering and organ regeneration techniques. Some of the new treatments that were not possible previously become reachable now. Because of the advent of surface chemistry, the CNT's biocompatibility has been significantly improved, making it possible to serve as tissue scaffolding materials to enhance the organ regeneration. The superior mechanic strength and chemical inert also makes it ideal for blood compatible applications, especially for cardiopulmonary bypass surgery. The applications of CNTs in these cardiovascular surgeries led to a remarkable improvement in mechanical strength of implanted catheters and reduced thrombogenicity after surgery. Moreover, the functionalized CNTs have been extensively explored for in vivo targeted drug or gene delivery, which could potentially improve the efficiency of many cancer treatments. However, just like other nanomaterials, the cytotoxicity of CNTs has not been well established. Hence, more extensive cytotoxic studies are warranted while converting the hydrophobic CNTs into biocompatible nanomaterials.

Development of immunosensors using carbon nanotubes.

With increasing reports on bioterrorism, avian flu, and other bio-threats, rapid and real time detection methods are highly warranted. Studies on developing highly sensitive immunosensors aiming at the early detection and clinical diagnoses of various diseases including cancer are undertaken all over the globe. Carbon nanotubes (CNTs) have been widely discussed as materials with enormous potential for a wide range of in vivo and in vitro bioapplications, ranging from drug delivery to highly sensitive biosensors, owing to their superior electronic and mechanical properties along with nanoscale dimensions. Though a lot of attention has been drawn toward carbon nanotubes for the past 15 years in academia and to a certain extent in industry, CNT-based immunosensors and other applications are still in the nascent stage, and there are many challenges to be overcome for the successful commercialization of the concepts. This article highlights on the recent developments and the possible impacts of carbon nanotube based immunosensors.