Biochemical Compositional Analysis of Cadaveric Rib Cartilage.

Background: Cadaveric rib is used as a cartilage source for reconstructive rhinoplasty in patients who lack sufficient native septal cartilage; however, these grafts are known to warp. Objective: To measure and compare the biochemical properties of cadaveric rib as related to age, gender, and cortical versus core location. Methods: Seven cadaveric rib cartilage specimens were obtained and sectioned into cortical and core segments. Biochemical assays were used to determine total collagen and sulfated glycosaminoglycan (sGAG) content. Results: Collagen was present in higher amounts in cortical segments than core samples (72.8 ± 35.14 vs. 37.3 ± 16.99 µg/mgww, p = 0.0005). sGAG was also shown to be more prevalent in cortical segments (25.47 ± 11.59 vs. 12.17 ± 7.15 µg/mgww, p < 0.0001). The concentrations of collagen and sGAG demonstrated a positive correlation (R2 = 0.44, p = 0.0004). Collagen and sGAG content decreased with the age of the donor (p = 0.001 and p < 0.0001, respectively), but donor gender did not appear to affect collagen or sGAG content (p = 0.62 and p = 0.43, respectively). Conclusion: Collagen and sGAG content was higher in cortical segments of cadaveric rib cartilage than in core segments, and higher in samples from younger cadavers as well.

Mechanical mechanism of rib spalling and sensitivity analysis of gangue parameters to rib spalling in gangue-bearing coal seams.

Rib spalling is one of the main factors restricting the safe and efficient production of the fully mechanized mining face in gangue-bearing coal seams, and the gangue has significant influence on the occurrence of rib spalling. In this study, the instability process and mechanical mechanism of rib spalling in gangue-bearing coal seams were studied, and the sensitivity of gangue parameters to rib spalling was analyzed. The simulation test of rib spalling under different gangue parameters was carried out by orthogonal tests. The width and depth of rib spalling were taken as evaluation indexes, and the influence of gangue parameters on the rib spalling was analyzed by variance analysis and significance tests. The results show that the failure process of rib spalling is characterized by the fracturing failure of the lower coal body, shear failure of the gangue layer, and the falling off of the upper coal body caused by the gravity; the gangue parameters (thickness, density, joint inclination, and internal friction angle) have an important influence on the sliding instability of the coal wall. In the sensitivity analysis, the influence of gangue parameters on the width of rib spalling is ordered as gangue density > joint inclination > gangue thickness > internal friction angle; the influence of gangue parameters on the depth of rib spalling is ordered as gangue density > joint inclination > internal friction angle > gangue thickness. Besides, the greater the gangue density, the less damage caused by stress concentration, and the lower the risk of rib spalling.

A single change in the aptamer of the Lactiplantibacillus plantarum rib operon riboswitch severely impairs its regulatory activity and leads to a vitamin B2 - overproducing phenotype.

Manufacturing of probiotics and functional foods using lactic acid bacteria (LAB) that overproduce vitamin B2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin-producing parental strain (UFG9) and a roseoflavin-resistant strain (B2) that carries a mutation in the FMN-aptamer of the potential rib operon riboswitch, were analysed for production and intra- and extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for LAB, exceeding by ~ 25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~ 8-fold higher in B2. Mutation B2 could decrease the stability of the aptamer's regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2-mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavin, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods.

Assessment of the ANDE 6C Rapid DNA system and investigative biochip for the processing of calcified and muscle tissue.

The ANDE 6C Rapid DNA system could offer a potential alternative for the processing of calcified and soft tissue samples, often encountered in mass disaster scenarios. While originally designed for single source buccal swabs, interest in the performance of these instruments when using other types of single source samples continues to grow. To enhance the recovery of otherwise lesser quality samples, the manufacturer developed the investigative biochip, an alternative to the NDIS approved Arrestee biochip for reference sample buccal swabs. This study explores the viability of using the ANDE 6C system and the investigative biochip to process soft and calcified tissue, and uses conventional sample processing to contrast the results. Though the success rate obtained using the instrument's expert system was lower than expected - 0% muscle, 11% ribs, and 50% teeth -, the ANDE 6C offers an advantage over conventional calcified tissue processing in terms of turn-around time and processing complexity. If robust analysis parameters can be established to allow the evaluation of the generated data by a qualified analyst on a third party software platform, the use of the ANDE 6C and investigative biochip could be a suitable alternative for currently employed procedures. However, as is the case with conventional DNA typing, the quantity, age, type of biological material and quality of the exemplars could all play a role in the success of the ANDE 6C typing process. In addition, it appears as if the calcified tissue pre-processing protocol that provides the better opportunity for the ANDE 6C success is not appropriate to be carried out in the field or by non-trained personnel as special equipment as well as a certain level of exe expertise and technique is necessary. Nevertheless, disaster victim and unidentified human remain samples could be processed in a laboratory setting using the Rapid DNA ANDE 6C platform provided sufficient material is available to conduct a second, 'rescue' sample processing if necessary.

Development of a quantitative PCR-based method for studying temporal DNA degradation in waterlogged bone.

While several studies have examined temporal DNA degradation in bones collected from terrestrial environments, studies on temporal DNA degradation in bones collected from aquatic environments are limited and mostly based on case studies. The objective of this study was to assess the impact of long-term submersion, aquatic environment, bone type and DNA extraction method on DNA quality and quantity. Bone samples (scapulae and ribs), collected every ~1000 ADD from a freshwater lake and river, underwent DNA extraction via ChargeSwitch® gDNA Plant Kit and organic phenol-chloroform methods, and DNA quantitation using both TaqMan and SYBR Green-based quantitative PCR (qPCR) methods. Results suggest that in both bone types, quality of recovered DNA (i.e., degradation index) declined significantly with increase in submersion time. Among two bone types, quality of recovered DNA from scapulae declined faster than rib samples. There was no significant difference in recovered DNA quantity between bone types, DNA extraction methods, or locations but various interactions between these variables showed significant difference. Overall, it can be concluded that DNA can be extracted from waterlogged bone in sufficient quantity to generate an STR profile up to 4000 ADD.

Drugs in bone: Detectability of substances of toxicological interest in different states of preservation.

In forensic contexts of advanced decomposition, when conventional matrices are no longer available for toxicological analyses, finding alternative matrices is necessary. The skeleton, which is fundamental for anthropologists and geneticists, could be useful also for toxicological purposes. The present study aims to examine what kind of information toxicological analysis performed on bones (the cranium and the ribs) in different states of preservation could provide to the forensic practitioner. Thirty cadavers with known pharmacological history, subjected to forensic autopsy at the Institute of Legal Medicine of Milan, were selected. Rib and cranium samples were collected from each body and separated into two parts in order to create two different states of preservation: One was cleaned from soft tissues and analyzed as a well-preserved bone sample; the other was submitted to a long maceration process, simulating complete skeletonization. All specimens were then processed with accelerated solvent extraction and the eluates analyzed using Q-Exactive™ Orbitrap™ Mass Spectrometer. The analysis of blood and skeletal matrices showed positive results for the tested substances in 63% of cases, mainly benzodiazepines, antidepressants, and drug abuse. Significant Pearson correlations were observed between non-macerated vs. macerated bone samples: r = 0.79 for rib samples, r = 0.61 for cranium samples, and r = 0.69 for all bone samples. As a consequence, the positive results confirm the potential of the bone tissue as an alternative matrix in forensic toxicology, even in cases of extremely decomposed bodies. This study also highlighted important elements for reconstructing the biological profile in cases of forensic anthropological concern.

Establishing a minimum PMI for bone sun bleaching in a UK environment with a controlled desert-simulated comparison.

Microenvironments play a significant part in understanding the post-mortem interval in forensic taphonomy. Recently, the value of weathering factors in relation to obtaining a PMI has been investigated further. In this study, observations were made to calculate the length of time it takes for three different bone elements (femur, rib, and scapula) to bleach in a UK summer and winter. This research also investigated whether there were any physicochemical modifications to the bone caused by bleaching. Porcine femora, scapulae, and ribs were placed into open and shaded areas of an outdoor research facility located in Oxfordshire, UK, during summer (July-Sep) and winter months (Dec-Mar). The specimens were monitored at 3-week intervals using photography, and an observational scoring method was developed to quantify the extent of bleaching. As temperatures are typically much lower in the UK compared with warmer climates, a controlled indoor-simulated desert experiment was also undertaken to be used as a control. This allowed sun bleaching and changes to the bone chemistry to be monitored in a controlled, high-UV environment for comparison with the UK outdoor experiments. Fourier transform infrared spectroscopy (FTIR) was employed to analyze physicochemical modifications to both the mineral and organic components of the bone. The FTIR was used to calculate crystallinity index (CI), mineral to organic ratio, and the relative amount of carbonate concentrations. Weather data was collected and a positive correlation was found between both ultraviolet (UV) levels and accumulated degree days (ADD) when compared with observational bleaching scores. Bleaching (whitening) of the bone samples occurred in both seasons but at different rates, with the bleaching process occurring at a slower rate in winter. During summer, the initial bleaching process was evident at 6 weeks, and by 9 weeks, the bones were an off-white colour. During the winter period, whitening of the bone started at 9 weeks; however, only the scapula and rib samples displayed a similar off-white colour. This colouration was observed at 13 weeks rather than at 9 weeks. The desert simulation samples started bleaching in a similar pattern to the outdoor samples after 1 week but the bones did not fully bleach. The bone chemistry, based on physicochemical properties obtained from the FTIR, showed a significant statistical difference between the simulated desert and winter season when compared against a control sample. For the winter samples, the mineral to organic ratio was significantly higher than that in the control, suggesting a reduction in the proportion of organic. For the samples in the simulated desert environment, the crystallinity index was significantly higher than that in the control samples, suggesting an increase in crystallinity. The results of this experiment support the fact that it is possible to achieve bleaching in a UK environment and that the minimal time frame for this to occur differs in seasons.

Locomotor rib kinematics in two species of lizards and a new hypothesis for the evolution of aspiration breathing in amniotes.

Most lizards walk and run with a sprawling gait in which the limbs are partly advanced by lateral undulation of the axial skeleton. Ribs and vertebrae are integral to this locomotor mode, but 3D motion of the axial skeleton has not been reported for lizard locomotion. Here, we use XROMM to quantify the relative motions of the vertebrae and ribs during slow treadmill locomotion in three savannah monitor lizards (Varanus exanthematicus) and three Argentine black and white tegus (Salvator merianae). To isolate locomotion, we selected strides with no concurrent lung ventilation. Rib rotations can be decomposed into bucket-handle rotation around a dorsoventral axis, pump-handle rotation around a mediolateral axis, and caliper rotations around a craniocaudal axis. During locomotion, every rib measured in both species rotated substantially around its costovertebral joint (8-17 degrees, summed across bucket, pump and caliper rotations). In all individuals from both species, the middle ribs rotated cranially through bucket and pump-handle motion during the propulsive phase of the ipsilateral forelimb. Axial kinematics during swing phase of the ipsilateral forelimb were mirror images of the propulsive phase. Although further work is needed to establish what causes these rib motions, active contraction of the hypaxial musculature may be at least partly responsible. Unilateral locomotor rib movements are remarkably similar to the bilateral pattern used for lung ventilation, suggesting a new hypothesis that rib motion during locomotion may have been an exaptation for the evolution of costal aspiration breathing in stem amniotes.

Diet and adult age-at-death among mobile foragers: A synthesis of bioarcheological methods.

OBJECTIVES: The research explores whether the combined study of cortical bone histology, bone morphology, and dietary stable isotopes can expand insights into past human health and adaptations, particularly dietary sufficiency and life span. MATERIALS AND METHODS: Midthoracic rib cortices from 54 South African Late Holocene adult skeletons (28 M, 24 F, two sex undetermined) are assessed by transmitted-light microscopy for cross-sectional area measurements, osteon area (On.Ar), osteon population density, and presence/absence of secondary osteon variants. Values for δ13 Cbone collagen , δ15 Nbone collagen , 14 C dates, Southwestern and Southern Cape geographic regions, body size measures, estimated ages-at-death from both morphological and histological methods are integrated into analyses, which include Spearman correlations, χ2 tests and Kruskal-Wallis ANOVAs. RESULTS: There is reduced On.Ar variability with higher δ15 N (r = -.41, p = .005); rib %cortical area and δ15 N are negatively correlated in the Southern Cape group (r = -.60, p = .03). Osteon variants are more common in older adults; histological ages at death are significantly older than those determined from gross morphology. DISCUSSION: We found bone tissue relationships with measures of diet composition, but indicators of dietary adequacy remain elusive. Relationships of tissue quality and isotopes suggest that some Southern Cape adults lived long lives. Osteon variants are associated with age-at-death; some association with diet remains possible. Gross morphological methods appear to underestimate adult ages-at-death, at least among small-bodied adults.

Development and validation of a method for analysing of duloxetine, venlafaxine and amitriptyline in human bone.

A method based on gas chromatography-mass spectrometry (GC-MS) is described for the determination of venlafaxine, amitriptyline and duloxetine in human bone. Pulverized samples were incubated in methanol for 1 h under ultrasonication, after the addition of sertraline as internal standard. The samples were centrifuged, and the supernatants were evaporated. Samples were then resuspended in 0.1 M phosphate buffer pH 6 and subjected to solid phase extraction. Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The assay was validated in the range 0.3-1 ng/mg (depending on the drug) to 500 ng/mg. The mean absolute recoveries ranged from 92.6% to 96.2%, the matrix effect from 76.9% to 103.3% and process efficiency from 74% to 95.9% depending on the analyte. The intra- and inter-assay accuracy values were always better than 20%. The validated method was then successfully applied to real bone samples from forensic cases in which toxicological analysis for these drugs in blood had been positive. Drugs were detected in bone in all blood positive results, the approximate concentrations being 36.4 ng/mg for amitriptyline, 19.3-3 ng/mg for duloxetine and 4.6-2 ng/mg for venlafaxine.

Alternative rib bone biopsy measurements to estimate changes in skeletal mineral reserves in cattle.

Rib bone biopsy samples are often used to estimate changes in skeletal mineral reserves in cattle but differences in sampling procedures and the bone measurements reported often make interpretation and comparisons among experiments difficult. 'Full-core' rib bone biopsy samples, which included the external cortical bone, internal cortical bone and trabecular bone (CBext, CBint and Trab, respectively), were obtained from cattle known to be in phosphorus (P) adequate (Padeq) or severely P-deficient (Pdefic) status. Experiments 1 and 2 examined growing steers and Experiment 3 mature breeder cows. The thickness of cortical bone, specific gravity (SG), and the amount and concentration of ash and P per unit fresh bone volume, differed among CBext, CBint and Trab bone. P concentration (mg/cc) was closely correlated with both SG and ash concentrations (pooled data, r=0.99). Thickness of external cortical bone (CBText) was correlated with full-core P concentration (FC-Pconc) (pooled data, r=0.87). However, an index, the amount of P in CBext per unit surface area of CBext (PSACB; mg P/mm2), was more closely correlated with the FC-Pconc (pooled data, FC-Pconc=37.0+146×PSACB; n=42, r=0.94, RSD=7.7). Results for measured or estimated FC-Pconc in 10 published studies with cattle in various physiological states and expected to be Padeq or in various degrees of Pdefic status were collated and the ranges of FC-Pconc indicative of P adequacy and P deficiency for various classes of cattle were evaluated. FC-Pconc was generally in the range 130 to 170 and 100 to 120 mg/cc fresh bone in Padeq mature cows and young growing cattle, respectively. In conclusion, the FC-Pconc could be estimated accurately from biopsy samples of CBext. This allows comparisons between studies where full-core or only CBext biopsy samples of rib bone have been obtained to estimate changes in the skeletal P status of cattle and facilitates evaluation of the P status of cattle.

Comparison of Different Swabs for Sampling Inorganic Gunshot Residue from Gunshot Wounds: Applicability and Reliability for the Determination of Firing Distance.

This study concentrates on samples of bare pork skin, with and without bristles, and dried bovine ribs shot with a semi-automatic pistol to find the best methodology and sampling surface in the search for inorganic gunshot residues (IGSR). Four quadrants of known surface areas were sampled at different distances from the bullet's hole with different swabs: tapes in graphite, Leukosilk® white tape, 3M® transparent tape, and a cotton swab to assess the technique able to collect the highest amounts of IGSR with the lowest contribution of the blank. The cotton swab wet in 10% HNO3 gave the best results. The highest amounts of IGSR, measured by ICP-OES and MS, were detected on a surface of 3-cm radius from the bullet's edge. The amount of metals collected decreased with the firing distance between 20 and 60 cm. The procedure was efficient for sampling different tissues like skin and bones.

90,000 year-old specialised bone technology in the Aterian Middle Stone Age of North Africa.

The question of cognitive complexity in early Homo sapiens in North Africa is intimately tied to the emergence of the Aterian culture (~145 ka). One of the diagnostic indicators of cognitive complexity is the presence of specialised bone tools, however significant uncertainty remains over the manufacture and use of these artefacts within the Aterian techno-complex. In this paper we report on a bone artefact from Aterian Middle Stone Age (MSA) deposits in Dar es-Soltan 1 cave on the Atlantic coast of Morocco. It comes from a layer that can be securely dated to ~90 ka. The typological characteristics of this tool, which suggest its manufacture and use as a bone knife, are comparatively similar to other bone artefacts from dated Aterian levels at the nearby site of El Mnasra and significantly different from any other African MSA bone technology. The new find from Dar es-Soltan 1 cave combined with those from El Mnasra suggest the development of a bone technology unique to the Aterian.

Protein-based forensic identification using genetically variant peptides in human bone.

Bone tissue contains organic material that is useful for forensic investigations and may contain preserved endogenous protein that can persist in the environment for extended periods of time over a range of conditions. Single amino acid polymorphisms in these proteins reflect genetic information since they result from non-synonymous single nucleotide polymorphisms (SNPs) in DNA. Detection of genetically variant peptides (GVPs) - those peptides that contain amino acid polymorphisms - in digests of bone proteins allows for the corresponding SNP alleles to be inferred. Resulting genetic profiles can be used to calculate statistical measures of association between a bone sample and an individual. In this study proteomic analysis on rib cortical bone samples from 10 recently deceased individuals demonstrates this concept. A straight-forward acidic demineralization protocol yielded proteins that were digested with trypsin. Tryptic digests were analyzed by liquid chromatography mass spectrometry. A total of 1736 different proteins were identified across all resulting datasets. On average, individual samples contained 454±121 (x¯±σ) proteins. Thirty-five genetically variant peptides were identified from 15 observed proteins. Overall, 134 SNP inferences were made based on proteomically detected GVPs, which were confirmed by sequencing of subject DNA. Inferred individual SNP genetic profiles ranged in random match probability (RMP) from 1/6 to 1/42,472 when calculated with European population frequencies in the 1000 Genomes Project, Phase 3. Similarly, RMPs based on African population frequencies were calculated for each SNP genetic profile and likelihood ratios (LR) were obtained by dividing each European RMP by the corresponding African RMP. Resulting LR values ranged from 1.4 to 825 with a median value of 16. GVP markers offer a basis for the identification of compromised skeletal remains independent of the presence of DNA template.

Evaluation of DNA Extraction Methods from Waterlogged Bones: A Pilot Study.

When deaths occur in water, soft tissue decomposes after a temperature-dependent period, making DNA identification dependent on bone. This study examined the effects of water on bone DNA purity and quantity, and determined the best of three extraction methods for isolating DNA. The organic phenol-chloroform method consistently extracted DNA approximating the accepted 260/280 purity value (~1.8); ChargeSwitch® gDNA Plant Kit and DNeasy Blood and Tissue Kit produced fair and unacceptable values, respectively. The purity value for humerus and rib samples was consistent across accumulated degree days (ADD). Significant differences in quantification among extraction methods and between bone types were identified. Ribs and ChargeSwitch® gDNA Plant Kit samples produced the lowest mean Ct values of the bone types and the extraction methods, respectively. Therefore, this study proposes that magnetic bead technology extraction methods and ribs be considered when processing bones that have been submerged in water for any length of time.

Citrate Content of Bone as a Measure of Postmortem Interval: An External Validation Study.

The postmortem interval (PMI) of skeletal remains is a crucial piece of information that can help establish the time dimension in criminal cases. Unfortunately, the accurate and reliable determination of PMI from bone continues to evade forensic investigators despite concerted efforts over the past decades to develop suitable qualitative and quantitative methods. A relatively new PMI method based on the analysis of citrate content of bone was developed by Schwarcz et al. The main objective of our research was to determine whether this work could be externally validated. Thirty-one bone samples were obtained from the Forensic Anthropology Center, University of Tennessee, Knoxville, and the Onondaga County Medical Examiner's Office. Results from analyzing samples with PMI greater than 2 years suggest that the hypothetical relationship between the citrate content of bone and PMI is much weaker than reported. It was also observed that the average absolute error between the PMI value estimated using the equation proposed by Schwarcz et al. and the actual ("true") PMI of the sample was negative indicating an underestimation in PMI. These findings are identical to those reported by Kanz et al. Despite these results this method may still serve as a technique to sort ancient from more recent skeletal cases, after further, similar validation studies have been conducted.

Scanning Electron Microscopy-Energy-Dispersive X-Ray (SEM/EDX): A Rapid Diagnostic Tool to Aid the Identification of Burnt Bone and Contested Cremains.

This study investigates the use of Scanning electron microscopy-energy-dispersive X-ray (SEM-EDX) as a diagnostic tool for the determination of the osseous origin of samples subjected to different temperatures. Sheep (Ovis aries) ribs of two experimental groups (fleshed and defleshed) were burned at temperatures of between 100°C and 1100°C in 100°C increments and subsequently analyzed with the SEM-EDX to determine the atomic percentage of present elements. Three-factor ANOVA analysis showed that neither the exposure temperature, nor whether the burning occurred with or without soft tissue present had any significant influence on the bone's overall elemental makeup (p > 0.05). The Ca/P ratio remained in the osseous typical range of between 1.6 and 2.58 in all analyzed samples. This demonstrates that even faced with high temperatures, the overall gross elemental content and atomic percentage of elements in bone remain stable, creating a unique "fingerprint" for osseous material, even after exposure to extreme conditions.

Evidence of preserved collagen in an Early Jurassic sauropodomorph dinosaur revealed by synchrotron FTIR microspectroscopy.

Fossilized organic remains are important sources of information because they provide a unique form of biological and evolutionary information, and have the long-term potential for genomic explorations. Here we report evidence of protein preservation in a terrestrial vertebrate found inside the vascular canals of a rib of a 195-million-year-old sauropodomorph dinosaur, where blood vessels and nerves would normally have been present in the living organism. The in situ synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectra exhibit the characteristic infrared absorption bands for amide A and B, amide I, II and III of collagen. Aggregated haematite particles (α-Fe2O3) about 6∼8 µm in diameter are also identified inside the vascular canals using confocal Raman microscopy, where the organic remains were preserved. We propose that these particles likely had a crucial role in the preservation of the proteins, and may be remnants partially contributed from haemoglobin and other iron-rich proteins from the original blood.

A test of the citrate method of PMI estimation from skeletal remains.

Citrate content in bone has been shown to be associated with the postmortem interval (PMI), with citrate decreasing after death as a function of time. Here we test this method using porcine ribs for the period of 1-165days after death, and also assess citrate content and variation from samples placed into two different postmortem environments (terrestrial and aquatic). Higher citrate variation, lower citrate recovery, and a weaker association with time were found in this study as compared to others. Citrate content, however, was found to decrease with increasing PMI, and the method was found to be easy and inexpensive to apply. No significant differences were found in citrate loss between terrestrial and aquatic environments. Although more research is needed, citrate content appears to be a promising new approach in estimating PMI from skeletal remains.