Optical photothermal infrared spectroscopy and discrete wavenumber imaging for high content screening of single cells.

Major drawbacks of direct mid-infrared spectroscopic imaging of single cells in an aqueous buffer are strong water absorption, low resolution typically above 10 µm, and Mie scattering effects. This study demonstrates how an indirect detection principle can overcome these drawbacks using the optical photothermal infrared (O-PTIR) technique for high-resolution discrete wavenumber imaging and fingerprint spectroscopy of cultivated cells as a model system in a simple liquid sample chamber. The O-PTIR spectra of six leukemia- and cancer-derived cell lines showed main IR bands near 1648, 1547, 1447, 1400, 1220, and 1088 cm-1. Five spectra of approximately 260 single cells per cell type were averaged, the O-PTIR data set was divided into leukemia-derived cells (THP-1, HL 60, Jurkat, and Raji) and cancer cells (HeLa and HepaRG), and partial least squares linear discriminant analysis (PLS-LDA) was applied in the spectral range 800-1800 cm-1 to train three classification models. A leukemia versus cancer cell model showed an accuracy of 90.0%, the HeLa versus HepaRG cell model had an accuracy of 95.4%, and the model for the distinction of leukemia cells had an accuracy of 75.4%. IR bands in linear discriminants (LDs) of the models were correlated with second derivative spectra that resolved more than 25 subbands. The IR and second derivative spectra of proteins, DNA, RNA and lipids were collected as references to confirm band assignments. O-PTIR images of single cells at a 200 nm step size were acquired at 1086, 1548, and 1746 cm-1 to visualize the nucleic acid, protein, and lipid distribution, respectively. Variations in subcellular features and in the lipid-to-protein and nucleic acid-to-protein ratios were identified that were consistent with biomolecular information in LDs. In conclusion, O-PTIR can provide high-quality spectra and images with submicron resolution of single cells in aqueous buffers that offer prospects in high-content screening applications.

Whole Liver Derived Acellular Extracellular Matrix for Bioengineering of Liver Constructs: An Updated Review.

Biomaterial templates play a critical role in establishing and bioinstructing three-dimensional cellular growth, proliferation and spatial morphogenetic processes that culminate in the development of physiologically relevant in vitro liver models. Various natural and synthetic polymeric biomaterials are currently available to construct biomimetic cell culture environments to investigate hepatic cell-matrix interactions, drug response assessment, toxicity, and disease mechanisms. One specific class of natural biomaterials consists of the decellularized liver extracellular matrix (dECM) derived from xenogeneic or allogeneic sources, which is rich in bioconstituents essential for the ultrastructural stability, function, repair, and regeneration of tissues/organs. Considering the significance of the key design blueprints of organ-specific acellular substrates for physiologically active graft reconstruction, herein we showcased the latest updates in the field of liver decellularization-recellularization technologies. Overall, this review highlights the potential of acellular matrix as a promising biomaterial in light of recent advances in the preparation of liver-specific whole organ scaffolds. The review concludes with a discussion of the challenges and future prospects of liver-specific decellularized materials in the direction of translational research.

Investigating biochemical and structural changes of glycated collagen using multimodal multiphoton imaging, Raman spectroscopy, and atomic force microscopy.

Advanced glycation end products (AGEs) form extracellular crosslinking with collagenous proteins, which contributes to the development of diabetic complications. In this study, AGEs-related pentosidine (PENT) crosslinks-induced structural and biochemical changes are studied using multimodal multiphoton imaging, Raman spectroscopy and atomic force microscopy (AFM). Decellularized equine pericardium (EP) was glycated with four ribose concentrations ranging between 5 and 200 mM and monitored for up to 30 days. Two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopic imaging probed elastin and collagen fibers, respectively. The glycated EP showed a decrease in the SHG intensities associated with loss of non-centrosymmetry of collagen and an increase of TPEF intensities associated with PENT crosslinks upon glycation. TPEF signals from elastin fibers were unaffected. A three-dimensional reconstruction with SHG + TPEF z-stack images visualized the distribution of collagen and elastin within the EP volume matrix. In addition, Raman spectroscopy (RS) detected changes in collagen-related bands and discriminated glycated from untreated EP. Furthermore, AFM scans showed that the roughness increases and the D-unit structure of fibers remained unchanged during glycation. The PENT crosslinked-induced changes are discussed in the context of previous studies of glutaraldehyde- and genipin-induced crosslinking and collagenase-induced digestion of collagen. We conclude that TPEF, SHG, RS, and AFM are effective, label-free, and non-destructive methods to investigate glycated tissues, differentiate crosslinking processes, and characterize general collagen-associated and disease-related changes, in particular by their RS fingerprints.

Etoposide-Entrapped Progesterone-Cationic Lipid Nanoaggregates as Selective Therapeutics against Etoposide-Resistant Colorectal Cancer Cells.

Drug resistance has a major impact on the treatment of several cancers. This is mainly due to the overexpression of cellular drug efflux proteins. Hence, drug-delivery systems that can avoid this resistance are needed. We report PR10, a progesterone-cationic lipid conjugate, as a self-assembling nanoaggregate that delivers a drug cargo of etoposide, a topoisomerase inhibitor, selectively to cancer cells. In this study, we observed that etoposide nanoaggregates (P : E) caused selective and enhanced toxicity in etoposide-resistant CT26 cancer cells (IC50 9 µM) compared to when etoposide (IC50 >20 µM) was used alone. Concurrently, no toxicity was observed in etoposide-sensitive HEK293 cells for P : E treatment (IC50 >20 µM). The P : E-treated cancer cells seem to have no effect on ABCB1 expression, but etoposide-treated cells exhibited a twofold increase in ABCB1 expression, a potent efflux protein for several xenobiotic compounds. This observation supports the notion that the enhanced toxicity of P : E nanoaggregates is due to their ability to keep the expression of ABCB1 low, thus allowing longer intracellular residence of etoposide. In a BALB/c orthotopic colorectal cancer model, the nanoaggregates led to enhanced survival (45 days) compared to etoposide-treated mice (39 days). These findings suggest that PR10 could be used as a potential cancer-selective etoposide delivery vehicle to treat several etoposide-resistant cancers with fewer side effects due to the nonspecific toxicity of the drug.

Epidemiology, Clinico-Pathological Characteristics, and Comorbidities of SARS-CoV-2-Infected Pakistani Patients.

SARS-CoV-2 is a causative agent for COVID-19 disease, initially reported from Wuhan, China. The infected patients experienced mild to severe symptoms, resulting in several fatalities due to a weak understanding of its pathogenesis, which is the same even to date. This cross-sectional study has been designed on 452 symptomatic mild-to-moderate and severe/critical patients to understand the epidemiology and clinical characteristics of COVID-19 patients with their comorbidities and response to treatment. The mean age of the studied patients was 58 ± 14.42 years, and the overall male to female ratio was 61.7 to 38.2%, respectively. In total, 27.3% of the patients had a history of exposure, and 11.9% had a travel history, while for 60% of patients, the source of infection was unknown. The most prevalent signs and symptoms in ICU patients were dry cough, myalgia, shortness of breath, gastrointestinal discomfort, and abnormal chest X-ray (p < 0.001), along with a high percentage of hypertension (p = 0.007) and chronic obstructive pulmonary disease (p = 0.029) as leading comorbidities. The complete blood count indicators were significantly disturbed in severe patients, while the coagulation profile and D-dimer values were significantly higher in mild-to-moderate (non-ICU) patients (p < 0.001). The serum creatinine (1.22 µmol L-1; p = 0.016) and lactate dehydrogenase (619 µmol L-1; p < 0.001) indicators were significantly high in non-ICU patients, while raised values of total bilirubin (0.91 µmol L-1; p = 0.054), C-reactive protein (84.68 mg L-1; p = 0.001), and ferritin (996.81 mg L-1; p < 0.001) were found in ICU patients. The drug dexamethasone was the leading prescribed and administrated medicine to COVID-19 patients, followed by remdesivir, meropenem, heparin, and tocilizumab, respectively. A characteristic pattern of ground glass opacities, consolidation, and interlobular septal thickening was prominent in severely infected patients. These findings could be used for future research, control, and prevention of SARS-CoV-2-infected patients.

Structural and Biochemical Changes in Pericardium upon Genipin Cross-Linking Investigated Using Nondestructive and Label-Free Imaging Techniques.

Tissue cross-linking represents an important and often used technique to enhance the mechanical properties of biomaterials. For the first time, we investigated biochemical and structural properties of genipin (GE) cross-linked equine pericardium (EP) using optical imaging techniques in tandem with quantitative atomic force microscopy (AFM). EP was cross-linked with GE at 37 °C, and its biochemical and biomechanical properties were observed at various time points up to 24 h. GE cross-linked EP was monitored by the normalized ratio between its second-harmonic generation (SHG) and two-photon autofluorescence emissions and remained unchanged for untreated EP; however, a decreasing ratio due to depleted SHG and elevated autofluorescence and a fluorescence band at 625 nm were found for GE cross-linked EP. The mean autofluorescence lifetime of GE cross-linked EP also decreased. The biochemical signature of GE cross-linker and shift in collagen bands were detected and quantified using shifted excitation Raman difference spectroscopy as an innovative approach for tackling artifacts with high fluorescence backgrounds. AFM images indicated a higher and increasing Young's modulus correlated with cross-linking, as well as collagen structural changes in GE cross-linked EP, qualitatively explaining the observed decrease in the second-harmonic signal. In conclusion, we obtained detailed information about the biochemical, structural, and biomechanical effects of GE cross-linked EP using a unique combination of optical and force microscopy techniques in a nondestructive and label-free manner.

Assessment of shifted excitation Raman difference spectroscopy in highly fluorescent biological samples.

Shifted excitation Raman difference spectroscopy (SERDS) can be used as an instrumental baseline correction technique to retrieve Raman bands in highly fluorescent samples. Genipin (GE) cross-linked equine pericardium (EP) was used as a model system since a blue pigment is formed upon cross-linking, which results in a strong fluorescent background in the Raman spectra. EP was cross-linked with 0.25% GE solution for 0.5 h, 2 h, 4 h, 6 h, 12 h, and 24 h, and compared with corresponding untreated EP. Raman spectra were collected with three different excitation wavelengths. For the assessment of the SERDS technique, the preprocessed SERDS spectra of two excitation wavelengths (784 nm-786 nm) were compared with the mathematical baseline-corrected Raman spectra at 785 nm excitation using extended multiplicative signal correction, rubberband, the sensitive nonlinear iterative peak and polynomial fitting algorithms. Whereas each baseline correction gave poor quality spectra beyond 6 h GE crosslinking with wave-like artefacts, the SERDS technique resulted in difference spectra, that gave superior reconstructed spectra with clear collagen and resonance enhanced GE pigment bands with lower standard deviation. Key for this progress was an advanced difference optimization approach that is described here. Furthermore, the results of the SERDS technique were independent of the intensity calibration because the system transfer response was compensated by calculating the difference spectrum. We conclude that this SERDS strategy can be transferred to Raman studies on biological and non-biological samples with a strong fluorescence background at 785 nm and also shorter excitation wavelengths which benefit from more intense scattering intensities and higher quantum efficiencies of CCD detectors.

Epidemiology, Clinico-Pathological Characteristics, and Comorbidities of SARS-CoV-2 infected Pakistani Patients

SARS-CoV-2 is a causative agent for COVI-19 disease, initially reported from Wuhan, China. Infected Patients experienced mild to severe symptoms, resulting in several fatalities due to a weak understanding of its pathogenesis, which is the same even to date. This cross-sectional study has been designed on four hundred and fifty-two symptomatic, mild-to-moderate, and severe/critical patients to understand the epidemiology and clinical characteristics of COVID-19 patients with their comorbidities and response to treatment. The mean age of studied patients was (58{+/-}14.42) years, and the overall male to female ratio was 61.7 to 38.2%, respectively. 27.3% of the patients had a history of exposure, 11.9% travel history, while for 60% of patients, the source of infection was unknown. The most prevalent signs and symptoms in ICU patients were dry coughs, myalgias, shortness of breath, gastrointestinal discomfort, and abnormal Chest X-ray (p<0.001), along with the high percentage of hypertension (p=0.007) and COPD (p=0.029) as leading comorbidities. Complete Blood Counts indicators were significantly increased in severe patients, while the Coagulation Profile and D-dimer values were significantly higher in mild-to-moderate (non-ICU) patients (p < 0.001). Serum Creatinine (1.22 umole L-1; p = 0.016) and LDH (619 umol L-1; p < 0.001) indicators were significantly high in non-ICU patients while, raised values of Total Bilirubin (0.91 umol L-1; p = 0.054), CRP (84.68 mg L-1; p = 0.001) and Ferritin (996.81 mg L-1; p < 0.001) were found in ICU patients. Drug Dexamethasone was the leading prescribed and administrated medicine to the COVID-19 patients, followed by Remdesivir, Meropenem, Heparin, and Tocilizumab, respectively. A characteristic pattern of Ground glass opacities (GGO), consolidation, and interlobular septal thickening were prominent in severely infected patients. These findings could be used for future research, control, and prevention of SARS-CoV-2 infected patients.

Contrasting effects of maize residue, coal gas residue and their biochars on nutrient mineralization, enzyme activities and CO2 emissions in sandy loess soil.

Mismanagement of crop straw and coal gas residue threatens the atmosphere and the economy. Nevertheless, thermal-pyrolysis is an option for management that turns bio-waste into biochar; its viability and adoption by the public as soil amendments is dependent on the agronomic and environmental values compared between biochar and the raw materials. We undertook a 60-day short-term analysis to assess the impact of various wastes and biochars, as well as inorganic nutrients (N), on carbon dioxide (CO2) fluxes, soil enzyme activities, soil fertility status, and microbial activities. There were eight treatments of soil amendments: without an amendment (CK), Nutrients (N), straw + nutrients (S+N), straw biochar + nutrients (SB+N), coal gas residue + nutrients (C+N), coal gas residue biochar + nutrients (CB+N), straw + straw biochar + nutrients (S+SB+N) and coal gas residue waste + coal gas residue biochar + nutrients (C+ CB +N). The results indicated that soil EC, pH, nitrate N (NO3 -- N), SOC, TN and available K were significantly (p < 0.05) increased coal gas residue biochar and combined with coal fly ash as compared to maize straw biochar and combined with maize straw and N treatments. The higher concentrations of soil MBC and MBN activities were increased in the maize straw application, while higher soil enzyme activity such as, invertase, urease and catalase were enhanced in the coal fly ash derived biochar treatments. The higher cumulative CO2 emissions were recorded in the combined applications of maize straw and its biochar as well as coal gas residue and its biochar treatment. Our study concludes, that maize straw and coal fly ash wastes were converted into biochar product could be a feasible substitute way of discarding, since land amendment and decreased CO2 fluxes and positive changes in soil microbial, and chemical properties, and can be confirmed under long-term conditions for reduction of economical and environment issues.

The Current Situation Regarding Long-Acting Insulin Analogues Including Biosimilars Among African, Asian, European, and South American Countries; Findings and Implications for the Future.

Background: Diabetes mellitus rates continue to rise, which coupled with increasing costs of associated complications has appreciably increased global expenditure in recent years. The risk of complications are enhanced by poor glycaemic control including hypoglycaemia. Long-acting insulin analogues were developed to reduce hypoglycaemia and improve adherence. Their considerably higher costs though have impacted their funding and use. Biosimilars can help reduce medicine costs. However, their introduction has been affected by a number of factors. These include the originator company dropping its price as well as promoting patented higher strength 300 IU/ml insulin glargine. There can also be concerns with different devices between the manufacturers. Objective: To assess current utilisation rates for insulins, especially long-acting insulin analogues, and the rationale for patterns seen, across multiple countries to inform strategies to enhance future utilisation of long-acting insulin analogue biosimilars to benefit all key stakeholders. Our approach: Multiple approaches including assessing the utilisation, expenditure and prices of insulins, including biosimilar insulin glargine, across multiple continents and countries. Results: There was considerable variation in the use of long-acting insulin analogues as a percentage of all insulins prescribed and dispensed across countries and continents. This ranged from limited use of long-acting insulin analogues among African countries compared to routine funding and use across Europe in view of their perceived benefits. Increasing use was also seen among Asian countries including Bangladesh and India for similar reasons. However, concerns with costs and value limited their use across Africa, Brazil and Pakistan. There was though limited use of biosimilar insulin glargine 100 IU/ml compared with other recent biosimilars especially among European countries and Korea. This was principally driven by small price differences in reality between the originator and biosimilars coupled with increasing use of the patented 300 IU/ml formulation. A number of activities were identified to enhance future biosimilar use. These included only reimbursing biosimilar long-acting insulin analogues, introducing prescribing targets and increasing competition among manufacturers including stimulating local production. Conclusions: There are concerns with the availability and use of insulin glargine biosimilars despite lower costs. This can be addressed by multiple activities.

Current utilization patterns for long-acting insulin analogues including biosimilars among selected Asian countries and the implications for the future.

INTRODUCTION: Prevalence rates for diabetes mellitus continue to rise, which, coupled with increasing costs of complications, has appreciably increased expenditure in recent years. Poor glycaemic control including hypoglycaemia enhances complication rates and associated morbidity, mortality and costs. Consequently, this needs to be addressed. Whilst the majority of patients with diabetes have type-2 diabetes, a considerable number of patients with diabetes require insulin to help control their diabetes. Long-acting insulin analogues were developed to reduce hypoglycaemia associated with insulin and help improve adherence, which can be a concern. However, their considerably higher costs have impacted on their funding and use, especially in countries with affordability issues. Biosimilars can help reduce the costs of long-acting insulin analogues thereby increasing available choices. However, the availability and use of long-acting insulin analogues can be affected by limited price reductions versus originators and limited demand-side initiatives to encourage their use. Consequently, we wanted to assess current utilisation rates for long-acting insulin analogues, especially biosimilars, and the rationale for patterns seen, across multiple Asian countries ranging from Japan (high-income) to Pakistan (lower-income) to inform future strategies. METHODOLOGY: Multiple approaches including assessing utilization and prices of insulins including biosimilars among six Asian countries and comparing the findings especially with other middle-income countries. RESULTS: Typically, there was increasing use of long-acting insulin analogues among the selected Asian countries. This was especially the case enhanced by biosimilars in Bangladesh, India, and Malaysia reflecting their perceived benefits. However, there was limited use in Pakistan due to issues of affordability similar to a number of African countries. The high use of biosimilars in Bangladesh, India and Malaysia was helped by issues of affordability and local production. The limited use of biosimilars in Japan and Korea reflects limited price reductions and demand-side initiatives similar to a number of European countries. CONCLUSIONS: Increasing use of long-acting insulin analogues across countries is welcomed, adding to the range of insulins available, which increasingly includes biosimilars. A number of activities are needed to enhance the use of long-acting insulin analogue biosimilars in Japan, Korea and Pakistan.

Foot and Mouth Disease (FMD) incidence in cattle and buffaloes and its associated farm-level economic costs in endemic India.

The study investigated the important epidemiological parameters and farm-level economic costs of FMD incidence in cattle and buffaloes during 2013-14 to 2015-16 in various states of India. Multistage random sampling procedure was adopted for the primary survey and data was collected through face-to-face personal interview from 18,609 cattle and buffalo rearing farm households from 123 districts across twelve states and one Union Territory. Besides epidemiological parameters, different farm-level direct and indirect loss associated with FMD was assessed at disaggregated level (states) by employing deterministic mathematical models. Highest number of affected villages and disease incidence was observed in non- FMD control programme (FMD-CP) implemented Madhya Pradesh and Assam states, respectively whereas negligible incidence was in FMD-CP implemented Punjab state. The disease incidence was high during 2013-14 and declined during 2014-15 and 2015-16, respectively implied severe incidence scenario (2013-14) succeeded by moderate (2014-15) and mild (2015-16) scenarios. The crossbred and high productive animals were severely affected than local breeds whereas on sexwise and agewise comparison revealed higher incidence in females and adult animals. During severe incidence scenario, milk loss/animal ranged from USD 6.87-47.44, 18.42-125.88, 16.33-91.43, and 27.17-123.62; mortality loss/animal ranged from USD 32.61-804.27, 30.76-577.7, 65.36-502.2, and 188.04-413.7; distress sale loss/animal ranged from USD 3.22-188.63, 64.34-519.3, 214.47-341.8, and 209.11-450.3; and opportunity cost of labour/animal from USD 5.49-54.29, 5.49-67.78; 7.95-31.37 and 9.83-72.38 in indigenous cattle, crossbred cattle, local and improved buffalo, respectively. The estimated draught power loss/animal varied from USD 39.46-142.94 with least being in Madhya Pradesh and highest in Assam states whereas the median treatment cost/animal was USD 9.18 and USD 27.07 in indigenous cattle and upgraded buffaloes, respectively. The total farm-level economic loss projected due to FMD in cattle and buffaloes in India was USD 3159 million (INR 221,110 million), USD 270 million (INR 18,910 million) and USD 152 million (INR 10,610 million), respectively during the severe, moderate and mild incidence scenarios at 2015-16 constant prices. The loss varied across the states, and in severe incidence scenario, the country might lose USD 3.2 billion/year and hence, the bi-annual vaccination schedule need to be strictly implemented in all the states. Besides timely vaccination coverage, managing unabated animal movement, educating and motivating the farmers to vaccinate their animals might reduce the incidence and consequential losses to various stakeholders in endemic states like India.

Monitoring Changes in Biochemical and Biomechanical Properties of Collagenous Tissues Using Label-Free and Nondestructive Optical Imaging Techniques.

We demonstrate the ability of nondestructive optical imaging techniques such as second-harmonic generation (SHG), two-photon fluorescence (TPF), fluorescence lifetime imaging (FLIM), and Raman spectroscopy (RS) to monitor biochemical and mechanical alterations in tissues upon collagen degradation. Decellularized equine pericardium (EP) was treated with 50 µg/mL bacterial collagenase at 37 °C for 8, 16, 24, and 32 h. The SHG ratio (defined as the normalized ratio between SHG and TPF signals) remained unchanged for untreated EP (stored in phosphate-buffered solution (PBS)), whereas treated EP showed a trend of a decreasing SHG ratio with increasing collagen degradation. In the fluorescence domain, treated EP experienced a red-shifted emission and the fluorescence lifetime had a trend of decreasing lifetime with increasing collagen digestion. RS monitors collagen degradation, the spectra had less intense Raman bands at 814, 852, 938, 1242, and 1270 cm-1. Non-negative least-squares (NNLS) modeling quantifies collagen loss and relative increase of elastin. The Young's modulus, derived from atomic force microscope-based nanoindentation experiments, showed a rapid decrease within the first 8 h of collagen degradation, whereas more gradual changes were observed for optical modalities. We conclude that optical imaging techniques like SHG, RS, and FLIM can monitor collagen degradation in a label-free manner and coarsely access mechanical properties in a nondestructive manner.

Effect of Monthly 100,000 IU Vitamin D Supplementation on Falls and Non-Vertebral Fractures.

Introduction Correlation between decreased levels of vitamin D in the blood of elderly patients and incidence of falls and fractures has been assessed in various studies; however, there is still ambiguity in data. In this study, we aim to establish the role of vitamin D supplements in minimizing the burden of falls and non-vertebral fractures in the elderly population in a local setting. Methods This single-blind, placebo-controlled randomized interventional study was conducted in the Internal Medicine Department of a tertiary care hospital in Pakistan from March 2018 to July 2020. Patients between the ages of 50 to 75 years were enrolled in the study and were randomly assigned to receive either placebo or 100,000 IU vitamin D oral tablets and were followed over 24 months, with regular follow-ups every three months. Results There was no significant difference in the probability of one or more falls for those assigned to the vitamin D group compared to those who received placebo (24.70% vs. 24.85%; hazard ratio [HR]: 0.99; 95% CI: 0.68-1.43). Similarly, the probability of non-vertebral fracture was also non-significant between both groups (4.7% vs. 5.7%; HR: 0.81; 95% CI: 0.32-2.01). Conclusion As per the results of this study, vitamin D supplementation had no beneficial effect on the reduction of falls and non-vertebral fractures in elderly patients. Further multi-center studies of longer duration are required to prove the favorable effects of vitamin D supplements.

Determination of the optimal cut-off value of serum prostate-specific antigen in the prediction of skeletal metastases on technetium-99m whole-body bone scan by receiver operating characteristic curve analysis.

Radionuclide whole-body bone scan is a useful investigation of choice to detect the skeletal metastases in prostate cancer. It is indicated in patients having elevated serum prostate-specific antigen (Sr. PSA) or patients with bone pain. Elevated Sr. PSA levels have high predictive value for skeletal metastases; however, there is no consensus regarding cut-off value of Sr. PSA above which bone scan is indicated. This study was performed to find out the accuracy of Sr. PSA test and to know the optimal cut-off value of Sr. PSA with high sensitivity and specificity in the prediction of skeletal metastases on bone scan in prostate cancer patients. A retrospective analysis of medical records of 307 prostate cancer patients referred to the department of nuclear medicine for bone scan between June 2009 and June 2014 was done. Of 307 patients, 15 cases were excluded due to nonavailability of Sr. PSA. Bone scan was performed 3 h after administration of 20 mCi Tc-99m methylene diphosphonate intravenously. Whole-body sweep imaging was performed and spot views were taken wherever required. Of 292 cases, 174 (59.58%) patients had positive bone scan for metastases and 118 (40.41%) patients had negative bone scan for metastases. Maximum and minimum Sr. PSA levels in positive and negative bone scan patients were 1260 and 0.02 ng/ml and 198.34 ng/ml and 0.01 ng/ml, respectively. On comparison of the mean Sr. PSA levels between positive and negative groups, we found significant Sr. PSA levels (P < 0.05). We used receiver operating characteristic (ROC) curve analyses to find out the accuracy of Sr. PSA test and to know the optimal cut-off value of Sr. PSA with maximum sensitivity and specificity in the prediction of skeletal metastases on bone scan. Area under ROC curve was 0.878 (87%). This indicates that the accuracy of Sr. PSA test in the prediction of skeletal metastases on bone scan was good. The optimal cut-off value of Sr. PSA in the prediction of positive bone scan for skeletal metastases in the management of prostate cancer was 29.16 ng/ml, with sensitivity and specificity of 89.0% and 74.6%, respectively. In this study, we conclude that the accuracy of Sr. PSA test in the prediction of skeletal metastases is good. ROC-derived optimal cut-off value of Sr. PSA for positive skeletal metastases on bone scan is >29.16 ng/ml; thus, the chances of getting positive bone scan for skeletal metastasis are less in prostate cancer patients with Sr. PSA <29.16 ng/ml. ROC-derived sensitivity and specificity of different possible cut-off points of Sr. PSA help reduce the false positive results and increase the diagnostic accuracy of bone scan in the detection of skeletal metastases in prostate cancer patients.

Association of Personal Protective Equipment with De Novo Headaches in Frontline Healthcare Workers during COVID-19 Pandemic: A Cross-Sectional Study.

OBJECTIVES: This study aimed to determine the association of personal protective equipment (PPE) usage with new-onset headaches and exacerbation of pre-existing headache disorders among healthcare workers at the frontlines during coronavirus disease 2019 (COVID-19) pandemic. MATERIALS AND METHODS: A descriptive cross-sectional survey was conducted across Pakistan in June-July 2020. The study was approved by Ethical Committee, Armed Forces Institute of Dentistry, Rawalpindi (IRB form no.905/Trg-ABP 1K2). A qualitative questionnaire was developed and was shared via different social networks. The questionnaire was closed when 241 responses were received. STATISTICAL ANALYSIS: Descriptive analysis was performed on demographic data. Chi-squared analysis was performed between demographic data and PPE-usage patterns among participants with or without de novo headaches. Univariable and multivariable logistic regression models were used to compare variables with the development of new-onset headaches. Chi-squared test was also performed between demographic data and other factors that may be causing new-onset headaches. A p-value < 0.05 was considered significant. RESULTS: A total of 241 healthcare workers participated, of which 68 participants (28.2%) reported de novo headaches since the start of the pandemic. Incidence of pre-existing headaches (odds ratio [OR] = 1.91; 95% confidence interval [CI]: 0.99-0.37; p = 0.049) was associated with new-onset headaches. Post hoc multivariable logistic regression analysis stated that incidence of pre-existing headaches (OR = 1.88; 95% CI: 0.94-3.78; p = 0.75) and age (OR = 2.21; 95% CI: 0.47-10.33; p = 0.36) was independently associated with new-onset PPE-induced headaches but was not statistically significant. Chi-squared analysis showed a statistically significant relationship between other factors (sleep deprivation, emotional stress, etc.) and department of activity, gender, and occupation (p < 0.05). CONCLUSION: Healthcare workers with previous history of pre-existing headaches were found to be more susceptible to PPE-induced headaches during COVID-19 pandemic. However, age and the department where the healthcare workers performed may also be risk factors.

FLIm and Raman Spectroscopy for Investigating Biochemical Changes of Bovine Pericardium upon Genipin Cross-Linking.

Biomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm-1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively.

Investigating Origins of FLIm Contrast in Atherosclerotic Lesions Using Combined FLIm-Raman Spectroscopy.

Background: Fluorescence lifetime imaging (FLIm) is a spectroscopic imaging technique able to characterize the composition of luminal surface of arterial vessels. Studies of human coronary samples demonstrated that distinct atherosclerotic lesion types are characterized by FLIm features associate with distinct tissue molecular makeup. While conventional histology has provided indications about potential sources of molecular contrast, specific information about the origin of FLIm signals is lacking. Here we investigate whether Raman spectroscopy, a technique able to evaluate chemical content of biological samples, can provide additional insight into the origin of FLIm contrast. Methods: Six human coronary artery samples were imaged using FLIm (355 nm excitation)-Raman spectroscopy (785 nm excitation) via a multimodal fiber optic probe. The spatial distribution of molecular contrast in FLIm images was analyzed in relationship with histological findings. Raman data was investigated using an endmember technique and compared with histological findings. A descriptive modeling approach based on multivariate regression was used to identify Raman bands related with changes in lifetime in four spectral channels (violet: 387/35 nm, blue: 443/29 nm, green: 546/38 nm, and red: 628/53 nm). Results: Fluorescence lifetime variations in the violet, blue and green spectral bands were observed for distinct areas of each tissue sample associated with distinct pathologies. Analysis of Raman signals from areas associated with normal, pathological intimal thickening, and fibrocalcific regions demonstrated the presence of hydroxyapatite, collagenous proteins, carotene, cholesterol, and triglycerides. The FLIm and Raman descriptive modeling analysis indicated that lifetime increase in the violet spectral band was associated with increased presence of cholesterol and carotenes, a new finding consistent with LDL accumulation in atherosclerotic lesions, and not with collagen proteins, as expected from earlier studies. Conclusions: The systematic, quantitative analysis of the multimodal FLIm-Raman dataset using a descriptive modeling approach led to the identification of LDL accumulation as the primary source of lifetime contrast in atherosclerotic lesions in the violet spectral range. Earlier FLIm validation studies relying on histopathological findings had associated this contrast to increased collagen content, also present in advanced lesions, thus demonstrating the benefits of alternative validation methods.

Open tendoachilles injuries due to squatting type of toilet seats: Five-year prospective observational study from a tertiary care center in India.

BACKGROUND: Open traumatic tendoachilles injuries due to toilet seats are least reported. The exact mechanism of such injuries is debatable. None of the studies have reported associated neurovascular injuries and the need for microvascular tissue transfer. METHODS: It is a 5-year prospective observational study from Sep 2013 - Aug 2018 at a tertiary care center on 26 patients who had sustained foot injuries due to squatting type toilet seats. All the patients were managed by thorough wound irrigation and debridement followed by repair of cut tendoachilles, other tendons and neurovascular structures. All the complications and secondary procedures required were recorded. Functional outcome was assessed by Boyden clinical outcome score. Follow up ranged from 1 to 5 years. RESULTS: All the 26 patients reported a particular mechanism of injury. Complete transection of tendoachilles was seen in 23 (88.5%) patients and partial transection in three (11.5%) patients. Microvascular repair of cut posterior tibial artery was undertaken in three and posterior tibial nerve in two cases and microvascular parascapular flap in one case for soft tissue reconstruction. Twenty-three (88.5%) patients had good to excellent Boyden score while three patients (11.5%) had fair to poor score at 1 year. Such severe injuries due to toilet seats have never been reported in literature. CONCLUSIONS: Squatting toilet seats can cause devastating foot injuries involving tendons and neurovascular structures and may require microvascular tissue transfer for definitive wound management. The risk of such injuries will continue unless some modifications are undertaken in the design of the seat.

FLIm-Guided Raman Imaging to Study Cross-Linking and Calcification of Bovine Pericardium.

Bovine pericardium (BP) is a vascular biomaterial used in cardiovascular surgery that is typically cross-linked for masking antigenicity and enhance stability. There is a need for biochemical evaluation of the tissue properties prior to implantation to ensure that quality and reliability standards are met. Here, engineered antigen removed BP (ARBP) that was cross-linked with 0.2% and 0.6% glutaraldehyde (GA), and further calcified in vitro to simulate graft calcifications upon implantation was characterized nondestructively using fluorescence lifetime imaging (FLIm) to identify regions of interest which were then assessed by Raman spectroscopy. We observed that the tissue fluorescence lifetime shortened, and that Raman bands at 856, 935, 1282, and 1682 cm-1 decreased, and at 1032 and 1627 cm-1 increased with increasing GA cross-linking. Independent classification analysis based on fluorescence lifetime and on Raman spectra discriminated between GA-ARBP and untreated ARBP with an accuracy of 91% and 66%, respectively. Pearson's correlation analysis showed a strong correlation between pyridinium cross-links measured with high-performance liquid chromatography and fluorescence lifetime measured at 380-400 nm (R = -0.76, p = 0.00094), as well as Raman bands at 856 cm-1 for hydroxy-proline (R = -0.68, p = 0.0056) and at 1032 cm-1 for hydroxy-pyridinium (R = 0.74, p = 0.0016). Calcified areas of GA cross-linked tissue showed characteristic hydroxyapatite (959 and 1038 cm-1) bands in the Raman spectrum and fluorescence lifetime shortened by 0.4 ns compared to uncalcified regions. FLIm-guided Raman imaging could rapidly identify degrees of cross-linking and detected calcified regions with high chemical specificity, an ability that can be used to monitor tissue engineering processes for applications in regenerative medicine.