Best Practices in Evolving Privacy Frameworks for Patient Age Data: Census Data Study.

BACKGROUND: Over the previous 4 decennial censuses, the population of the United States has grown older, with the proportion of individuals aged at least 90 years old in the 2010 census being more than 2 and a half times what it was in the 1980 census. This suggests that the threshold for constraining age introduced in the Safe Harbor method of the HIPAA (Health Insurance Portability and Accountability Act) in 1996 may be increased without exceeding the original levels of risk. This is desirable to maintain or even increase the utility of affected data sets without compromising privacy. OBJECTIVE: In light of the upcoming release of 2020 census data, this study presents a straightforward recipe for updating age-constrained thresholds in the context of new census data and derives recommendations for new thresholds from the 2010 census. METHODS: Using census data dating back to 1980, we used group size considerations to analyze the risk associated with various maximum age thresholds over time. We inferred the level of risk of the age cutoff of 90 years at the time of HIPAA's inception in 1996 and used this as a baseline from which to recommend updated cutoffs. RESULTS: The maximum age threshold may be increased by at least 2 years without exceeding the levels of risk conferred in HIPAA's original recommendations. Moreover, in the presence of additional information that restricts the population in question to a known subgroup with increased longevity (for example, restricting to female patients), the threshold may be increased further. CONCLUSIONS: Increasing the maximum age threshold would enable the data user to gain more utility from the data without introducing risk beyond what was originally envisioned with the enactment of HIPAA. Going forward, a recurring update of such thresholds is advised, in line with the considerations detailed in the paper.

Participation in patient support forums may put rare disease patient data at risk of re-identification.

BACKGROUND: Rare disease patients often struggle to find both medical advice and emotional support for their diagnosis. Consequently, many rare disease patient support forums have appeared on hospital webpages, social media sites, and on rare disease foundation sites. However, we argue that engagement in these groups may pose a healthcare data privacy threat to many participants, since it makes a series of patient indirect identifiers 'readily available' in combination with rare disease conditions. This information produces a risk of re-identification because it may allow a motivated attacker to use the unique combination of a patient's identifiers and disease condition to re-identify them in anonymized data. RESULTS: To assess this risk of re-identification, patient direct and indirect identifiers were mined from patient support forums for 80 patients across eight rare diseases. This data mining consisted of scanning patient testimonials, social media sites, and public records for the collection of identifiers linked to a rare disease patient. The number of people in the United States that may share each patient's combination of marital status, 3-digit ZIP code, age, and sex, as well as their rare disease condition, was then estimated, as such information is commonly found in health records which have undergone de-identification by HIPAA's 'Safe Harbor.' The study showed that by these estimations, nearly 75% of patients could be at high risk for re-identification in healthcare datasets in which they appear, due to their unique combination of identifiers. CONCLUSIONS: The results of this study show that these rare disease patients, due to their choice to provide support for their community, are putting all their healthcare data at risk of re-identification. This paper demonstrates how simple adjustments to participation guidelines in such support forums, in combination with improved privacy measures at the organizational level, could mitigate this risk of re-identification. Additionally, this paper suggests the potential for future investigation into consideration of certain 'risky' International Classification of Diseases (ICD) codes as quasi-identifiers in de-identified datasets to further protect patients' privacy, while maintaining the utility of such rare disease support groups.

Identification of C-ß-d-Glucopyranosyl Azole-Type Inhibitors of Glycogen Phosphorylase That Reduce Glycogenolysis in Hepatocytes: In Silico Design, Synthesis, in Vitro Kinetics, and ex Vivo Studies.

Several C-ß-d-glucopyranosyl azoles have recently been uncovered as among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Toward further exploring their translational potential, ex vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the molecular mechanics-generalized Born surface area (MM-GBSA) method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson (RP) and Spearman (RS) correlations (both 0.98), predictive index (PI = 0.99), and area under the receiver operating characteristic curve (AU-ROC = 0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using density functional theory (DFT). Seven 2-aryl-4(5)-(ß-d-glucopyranosyl)-imidazoles and 2-aryl-4-(ß-d-glucopyranosyl)-thiazoles were subsequently synthesized, and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best Ki values in the low micromolar range (5c = 1.97 µM; 13b = 4.58 µM). Ten C-ß-d-glucopyranosyl azoles were then tested ex vivo in mouse primary hepatocytes. Four of these (5a-c and 9d) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC50 = 30-60 µM). Structural and predicted physicochemical properties associated with their effectiveness were analyzed with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring, and the calculated pKa (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favored, while within the cell, there is predicted more favorable binding to GP in the protonated form.

A multidisciplinary study of 3-(ß-d-glucopyranosyl)-5-substituted-1,2,4-triazole derivatives as glycogen phosphorylase inhibitors: Computation, synthesis, crystallography and kinetics reveal new potent inhibitors.

3-(ß-d-Glucopyranosyl)-5-substituted-1,2,4-triazoles have been revealed as an effective scaffold for the development of potent glycogen phosphorylase (GP) inhibitors but with the potency very sensitive to the nature of the alkyl/aryl 5-substituent (Kun et al., Eur. J. Med. Chem. 2014, 76, 567). For a training set of these ligands, quantum mechanics-polarized ligand docking (QM-PLD) demonstrated good potential to identify larger differences in potencies (predictive index PI = 0.82) and potent inhibitors with Ki's < 10 µM (AU-ROC = 0.86). Accordingly, in silico screening of 2335 new analogues exploiting the ZINC docking database was performed and nine predicted candidates selected for synthesis. The compounds were prepared in O-perbenzoylated forms by either ring transformation of 5-ß-d-glucopyranosyl tetrazole by N-benzyl-arenecarboximidoyl chlorides, ring closure of C-(ß-d-glucopyranosyl)formamidrazone with aroyl chlorides, or that of N-(ß-d-glucopyranosylcarbonyl)arenethiocarboxamides by hydrazine, followed by deprotections. Kinetics experiments against rabbit muscle GPb (rmGPb) and human liver GPa (hlGPa) revealed five compounds as potent low µM inhibitors with three of these on the submicromolar range for rmGPa. X-ray crystallographic analysis sourced the potency to a combination of favorable interactions from the 1,2,4-triazole and suitable aryl substituents in the GP catalytic site. The compounds also revealed promising calculated pharmacokinetic profiles.

Plastic waste sacks alter the rate of decomposition of dismembered bodies within.

As a result of criminal activity, human bodies are sometimes dismembered and concealed within sealed, plastic waste sacks. Consequently, due to the inhibited ingress of insects and dismemberment, the rate of decomposition of the body parts within may be different to that of whole, exposed bodies. Correspondingly, once found, an estimation of the postmortem interval may be affected and lead to erroneous inferences. This study set out to determine whether insects were excluded and how rate of decomposition was affected inside such plastic sacks. The limbs, torsos and heads of 24 dismembered pigs were sealed using nylon cable ties within plastic garbage sacks, half of which were of a type claimed to repel insects. Using a body scoring scale to quantify decomposition, the body parts in the sacks were compared to those of ten exposed, whole pig carcasses. Insects were found to have entered both types of plastic sack. There was no difference in rate of decomposition in the two types of sack (F 1,65 = 1.78, p = 0.19), but this was considerably slower than those of whole carcasses (F 1,408 = 1453, p < 0.001), with heads showing the largest differences. As well as a slower decomposition, sacks resulted in formation of some adipocere tissue as a result of high humidity within. Based upon existing methods, postmortem intervals for body parts within sealed sacks would be significantly underestimated.

The distribution of blow fly (Diptera: Calliphoridae) larval lengths and its implications for estimating post mortem intervals.

The length or stage of development of blow fly (Diptera: Calliphoridae) larvae may be used to estimate a minimum postmortem interval, often by targeting the largest individuals of a species in the belief that they will be the oldest. However, natural variation in rate of development, and therefore length, implies that the size of the largest larva, as well as the number of larvae longer than any stated length, will be greater for larger cohorts. Length data from the blow flies Protophormia terraenovae and Lucilia sericata were collected from one field-based and two laboratory-based experiments. The field cohorts contained considerably more individuals than have been used for reference data collection in the literature. Cohorts were shown to have an approximately normal distribution. Summary statistics were derived from the collected data allowing the quantification of errors in development time which arise when different sized cohorts are compared through their largest larvae. These errors may be considerable and can lead to overestimation of postmortem intervals when making comparisons with reference data collected from smaller cohorts. This source of error has hitherto been overlooked in forensic entomology.

An Improved Equation for TBS and ADD: Establishing a Reliable Postmortem Interval Framework for Casework and Experimental Studies.

Megyesi et al.'s (J Forensic Sci, 2005, 50, 618) paper was important to forensic anthropology as it introduced a quantitative framework for estimating time since death in human cadavers, based upon physical appearance by way of scoring on a novel scale. However, errors concerning rounding, temperature scale, and incorrect use of a statistical regression model render their predictive formula unusable. Based upon only their more reliable data, a more appropriate regression model to predict accumulated degree days (ADD) from total body score (TBS) is presented. The new model is also a superior fit (r(2)  = 0.91) and produces markedly narrower confidence intervals than the original, which also allowed impossible, negative ADD values. Explanations of the shortcomings in the original analysis and calculations are presented, which it is hoped will help forensic scientists avoid making similar mistakes.

An evaluation of indirubin analogues as phosphorylase kinase inhibitors.

Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6]). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170-0.360µM, with indirubin-3'-acetoxime (1c) the most potent. 7-Bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20]) is revealed as a specific inhibitor of PhK (IC50=1.8µM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/ß), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work.

Decomposition Rate and Pattern in Hanging Pigs.

Accurate prediction of the postmortem interval requires an understanding of the decomposition process and the factors acting upon it. A controlled experiment, over 60 days at an outdoor site in the northwest of England, used 20 freshly killed pigs (Sus scrofa) as human analogues to study decomposition rate and pattern. Ten pigs were hung off the ground and ten placed on the surface. Observed differences in the decomposition pattern required a new decomposition scoring scale to be produced for the hanging pigs to enable comparisons with the surface pigs. The difference in the rate of decomposition between hanging and surface pigs was statistically significant (p=0.001). Hanging pigs reached advanced decomposition stages sooner, but lagged behind during the early stages. This delay is believed to result from lower variety and quantity of insects, due to restricted beetle access to the aerial carcass, and/or writhing maggots falling from the carcass.

The Effect of Clothing on the Rate of Decomposition and Diptera Colonization on Sus scrofa Carcasses.

Twenty Sus scrofa carcasses were used to study the effect the presence of clothing had on decomposition rate and colonization locations of Diptera species; 10 unclothed control carcasses were compared to 10 clothed experimental carcasses over 58 days. Data collection occurred at regular accumulated degree day intervals; the level of decomposition as Total Body Score (TBSsurf ), pattern of decomposition, and Diptera present was documented. Results indicated a statistically significant difference in the rate of decomposition, (t427  = 2.59, p = 0.010), with unclothed carcasses decomposing faster than clothed carcasses. However, the overall decomposition rates from each carcass group are too similar to separate when applying a 95% CI, which means that, although statistically significant, from a practical forensic point of view they are not sufficiently dissimilar as to warrant the application of different formulae to estimate the postmortem interval. Further results demonstrated clothing provided blow flies with additional colonization locations.

National characteristics and variation in Arabic handwriting.

From each of four Arabic countries; Morocco, Tunisia, Jordan and Oman, 150 participants produced handwriting samples which were examined to assess whether national characteristics were discernible. Ten characters, which have different configurations depending upon their position in the word, along with one short word, were classified into distinguishable forms, and these forms recorded for each handwriting sample. Tests of independence showed that character forms used were not independent of country (p<0.001) for all but one character-position (this was dropped from subsequent analyses). A correspondence analysis ordination plot and analysis of similarity (R=0.326, p=0.0002) showed that whole samples were discernibly grouped by country, and a tree analysis produced a classification which was 71% accurate for the original data and 83% accurate for 80 new handwriting samples that underwent 'blind' classification. When the countries were combined into two regions, North Africa and Middle East, the grouping was more marked. Thus, there appears to be some scope for narrowing down the nationality, and particularly the wider geographical region of an author based upon the character forms they use in Arabic handwriting.

Quantifying the temperature of maggot masses and its relationship to decomposition.

Numerous Calliphoridae species have been observed to form larval aggregations during the feeding stage of development, resulting in localized increases in temperature. This study investigates the relationship between maggot numbers in a mass and heat generation. Single-species aggregations (Lucilia sericata) of various sizes (50-2500 individuals) were reared in the laboratory at a constant ambient temperature of 22°C. Internal mass temperatures were recorded every 5 min throughout the feeding stage of development. Results showed that mass temperatures increased with mass numbers (p-value < 0.001), ranging from 2.5 to 14°C above ambient. A minimum mass size of 1200 produced overall temperatures that were significantly warmer than ambient, diverging away from 22°C after c. 26 h. These results indicate that the microclimate of a mass has the potential to differ significantly from ambient, which may be influencing larval development rates and should therefore be factored into mPMI estimates to increase accuracy.

A preliminary examination of differential decomposition patterns in mass graves.

Five pairs of mass graves, each containing carcasses of 21 rabbits, were used to examine differential decomposition at four locations within the burial: surface, deep, mid-outer, and core. Every 100 accumulated degree days (ADD), a pair of graves was exhumed, and total body score (TBS) and internal carcass temperature of each rabbit were recorded. Decomposition did not differ for core- and deep-positioned carcasses (p = 0.13); differences were significant (p < 0.001) for all other location comparisons. Decomposition occurred fastest in shallow carcasses, followed by mid-outer carcasses; deep and core carcasses decomposed slowest and at rates not significantly different from one another. Adipocere formation was minimal and confined to deep carcasses. Carcass location within the mass grave significantly influenced internal carcass temperature (p < 0.001); a mean internal temperature difference of ca. 1°C existed between deep and shallow carcasses (30 cm apart). Effects of mass compactness and oxygenation require further investigation.

Development of a multiplex system to assess DNA persistence in taphonomic studies.

In this study, we have developed a PCR multiplex that can be used to assess DNA degradation and at the same time monitor for inhibition: primers have been designed to amplify human, pig, and rabbit DNA, allowing pig and rabbit to be used as experimental models for taphonomic research, but also enabling studies on human DNA persistence in forensic evidence. Internal amplified controls have been added to monitor for inhibition, allowing the effects of degradation and inhibition to be differentiated. Sequence data for single-copy nuclear recombination activation gene (RAG-1) from human, pig, and rabbit were aligned to identify conserved regions and primers were designed that targeted amplicons of 70, 194, 305, and 384 bp. Robust amplification in all three species was possible using as little as 0.3 ng of template DNA. These have been combined with primers that will amplify a bacterial DNA template within the PCR. The multiplex has been evaluated in a series of experiments to gain more knowledge of DNA persistence in soft tissues, which can be important when assessing what material to collect following events such as mass disasters or conflict, when muscle or bone material can be used to aid with the identification of human remains. The experiments used pigs as a model species. When whole pig bodies were exposed to the environment in Northwest England, DNA in muscle tissue persisted for over 24 days in the summer and over 77 days in the winter, with full profiles generated from these samples. In addition to time, accumulated degree days (ADD) were also used as a measure that combines both time and temperature-24 days was in summer equivalent to 295 ADD whereas 77 days in winter was equivalent to 494 ADD.

N-alkylated 2,3,3-trimethylindolenines and 2-methylbenzothiazoles. Potential lead compounds in the fight against Saccharomyces cerevisiae infections.

The synthesis of a variety of N-alkylated 2,3,3-trimethylindolenines and 2-methylbenzothiazoles is reported herein. Their potential as antifungal agents is evaluated by preliminary screening against Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe), and Candida albicans (C. albicans). Statistical analyses illustrate a strong relationship between chain length and growth inhibition for S. cerevisiae and S. pombe (p < 0.0001 in every case). Of particular interest is the activity of both sets of compounds against S. cerevisiae, as this is emerging as an opportunistic pathogen, especially in immunosuppressed and immunocompromised patients. Bioassays were set up to compare the efficacy of our range of N-alkylated compounds against classic antifungal agents; Amphotericin B and Thiabendazole.

Differential decomposition patterns in charred versus un-charred remains.

Although researchers have examined many aspects of fire modification, the rate and pattern of decomposition in charred remains have not been studied previously. This study utilized 48 domestic pigs, divided into 24 charred (head, neck, and limbs burned to Crow-Glassman level 1 and torso to level 2) and 24 un-charred pig carcasses. Decomposition of control carcasses was scored at 50 accumulated degree days (ADD) intervals, and charred carcasses were also observed and photographed at this time. A Charred Body Scale was subsequently created, and charred carcasses were scored retrospectively for the same ADD intervals. Analysis using a mixed-effect repeated measures model indicated that, while decomposition rate was not statistically different between the two groups (p = 0.2692), the charred remains initially displayed an ostensibly more advanced pattern. Body regions displaying significant charring decomposed at a faster rate (p < 0.001), while areas with very light levels of charring decomposed at a significantly slower rate (p < 0.001).

The decomposition of rabbit carcasses and associated necrophagous Diptera in Kuwait.

Rabbit carcasses were used to compare rates of decomposition and associated assemblages of Diptera at four discernable habitat types in Kuwait; a country of a region with a paucity of such reference data. Carcasses in an urban habitat showed faster decomposition (as measured by percentage weight loss) than in agricultural, coastal or desert habitats, even with accumulated degree days (ADD) as the explanatory variable (t=2.73, df=34, p=0.010) to compensate for temperature differences. Taxa of Diptera at the four habitats became more similar as decomposition progressed, suggesting such differences between habitats were not marked. The occurrence of Chrysomyia megacephala and Lucilia sericata had not previously been recorded in Kuwait.

The formation of early stage adipocere in submerged remains: a preliminary experimental study.

In some circumstances, the presence of adipocere may retard decomposition and complicate postmortem interval estimation. This article explores the correlation between Accumulated Degree Days (ADD) and early stage formation of adipocere. Sixty wild rabbit (Oryctolagus cuniculus) carcasses were used in this experiment; a control group (N = 30) deposited directly on the ground surface and an experimental group (N = 30) completely submersed in water in individual buckets. Data (water and inner body temperature, pH, and total body score) were collected every 100 ADD. Results indicated that early stage adipocere is correlated to ADD and that its formation on submersed remains is more likely to occur after 630 ADD. Skin sloughing promoted the formation of adipocere. No adipocere was formed on any of the control group rabbits. This study also highlights the fact that multiple factors influence adipocere formation and it is suggested that further research needs to be conducted into this area.

The influence of insects on decomposition rate in buried and surface remains.

This article reports results of a comparative study of decomposition rates of wild rabbit (Oryctolagus cuniculus) which were either (i) buried after exposure to insect activity, (ii) buried without exposure, (iii) kept above ground behind an insect screen, or (iv) continuously exposed above ground in a field experiment. Results showed that dipteran oviposition occurred consistently in groups i and iv only. Decomposition rates (measured by Total Body Score every c. 50 accumulated degree days [ADD]) of rabbits kept behind the screen and those buried without exposure showed no difference (p = 0.450). This was significantly slower than those buried after exposure (p = 0.0016) which was in turn significantly slower than those continuously exposed (p << 0.001). Temperatures collected from animals showed the presence of feeding larvae increased intra-abdominal temperatures to >5 degrees C above ambient. The findings support the assertion that insect presence is the primary agent affecting decomposition rate via tissue consumption and also the heat they generate.