A plasma metabolite panel as biomarkers for early primary breast cancer detection.

In recent years, metabolites have attracted substantial attention as promising novel biomarkers of various diseases. However, breast cancer plasma metabolite studies are still in their infancy. Here, we investigated the potential of metabolites to serve as minimally invasive, early detection markers of primary breast cancer. We profiled metabolites extracted from the plasma of primary breast cancer patients and healthy controls using tandem mass spectrometry (UHPLC-MS/MS and FIA-MS/MS). Two metabolites were found to be upregulated, while 16 metabolites were downregulated in primary breast cancer patients compared to healthy controls in both the training and validation cohorts. A panel of seven metabolites was selected by LASSO regression analysis. This panel could differentiate primary breast cancer patients from healthy controls, with an AUC of 0.87 (95% CI: 0.81 ~ 0.92) in the training cohort and an AUC of 0.80 (95% CI: 0.71 ~ 0.87) in the validation cohort. These significantly differentiated metabolites are mainly involved in the amino acid metabolism and breast cancer cell growth pathways. In conclusion, using a metabolomics approach, we identified metabolites that have potential value for development of a multimarker blood-based test to complement and improve early breast cancer detection. The panel identified herein might be part of a prescreening tool, especially for younger women or for closely observing women with certain risks, to facilitate decision making regarding which individuals should undergo further diagnostic tests. In the future, the combination of metabolites and other blood-based molecular marker sets, such as DNA methylation, microRNA, and cell-free DNA mutation markers, will be an attractive option.

Serum very long-chain fatty acid-containing lipids predict response to immune checkpoint inhibitors in urological cancers.

Checkpoint inhibitors (CPI) have significantly changed the therapeutic landscape of oncology. We adopted a non-invasive metabolomic approach to understand immunotherapy response and failure in 28 urological cancer patients. In total, 134 metabolites were quantified in patient sera before the first, second, and third CPI doses. Modeling the association between metabolites and CPI response and patient characteristics revealed that one predictive metabolite class  (n = 9/10) were very long-chain fatty acid-containing lipids (VLCFA-containing lipids). The best predictive performance was achieved through a multivariate model, including age and a centroid of VLCFA-containing lipids prior to first immunotherapy (sensitivity: 0.850, specificity: 0.825, ROC: 0.935). We hypothesize that the association of VLCFA-containing lipids with CPI response is based on enhanced peroxisome signaling in T cells, which results in a switch to fatty acid catabolism. Beyond use as a novel predictive non-invasive biomarker, we envision that nutritional supplementation with VLCFA-containing lipids might serve as an immuno sensitizer.

Reduced mRNA expression levels of NFE2L2 are associated with poor outcome in breast cancer patients.

BACKGROUND: The transcription factor nuclear factor erythroid 2-related factor 2 (NFE2L2; previously known as NRF2) is a crucial regulator of the intracellular antioxidant response. It controls the expression of genes involved in the detoxification and elimination of reactive oxidants and electrophilic agents. The role of NFE2L2 in cancer is subject of controversial discussion, as it has been reported to have both pro-and anti-tumourigenic functions. To shed some light on this paradox, we analysed the NFE2L2 mRNA expression levels in breast cancer and its association with clinicopathological features and survival. METHODS: We retrospectively evaluated the NFE2L2 mRNA expression levels in tumour tissue of two independent breast cancer patient cohorts. In the training set we analysed data from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). In the test set we measured the NFE2L2 mRNA expression levels in 176 breast tumour tissues by quantitative real-time reverse transcription PCR (qRT-PCR). Group differences were analysed using Mann-Whitney U-test, and associations between NFE2L2 mRNA expression levels and clinicopathological features were examined by means of univariate and multivariate survival analyses. Furthermore, we compared NFE2L2 mRNA expression levels between tumour and normal breast tissue samples by means of 108 paired samples from the The Cancer Genome Atlas (TCGA) dataset. RESULTS: In the training set we identified an independent predictive value for high NFE2L2 mRNA expression levels [HRdisease specific death 0.8 (0.6-1.0), P = 0.041; HRdeath 0.8 (0.6-1.0), P = 0.023] especially in the subgroup of oestrogen receptor (ER) positive tumours [HRdisease specific death 0.6 (0.4-0.9), P = 0.008; HRdeath 0.6 (0.4-0.8), P = 0.001]. Similarly, we found this association also in the test set [HRrelapse 0.4 (0.2-0.9), P = 0.031] and again, more pronounced in patients with ER positive tumours [HRrelapse 0.2 (0.1-0.7), P = 0.012]. In addition, we observed generally lower NFE2L2 expression levels in tumour tissues than in normal breast tissues. CONCLUSION: We concluded that reduced NFE2L2 mRNA expression in tumour tissues is an independent predictor of shortened survival in breast cancer patients.

Association of TNFRSF10D DNA-methylation with the survival of melanoma patients.

In this retrospective pilot study, the DNA-methylation status of genes that have been demonstrated to be involved in melanoma carcinogenesis was analyzed in order to identify novel biomarkers for the risk assessment of melanoma patients. We analyzed DNA extracted from punch-biopsies from 68 formalin-fixed paraffin-embedded (FFPE) melanoma specimens. Using MethyLight PCR, we examined 20 genes in specimens from a training set comprising 36 melanoma patients. Selected candidate genes were validated in a test set using FFPE tissue samples from 32 melanoma patients. First, we identified the TNFRSF10D DNA-methylation status (TNFRSF10D methylated vs. unmethylated) as a prognostic marker for overall (p = 0.001) and for relapse-free survival (p = 0.008) in the training set. This finding was confirmed in the independent test set (n = 32; overall survival p = 0.041; relapse-free survival p = 0.012). In a multivariate Cox-regression analysis including all patients, the TNFRSF10D DNA-methylation status remained as the most significant prognostic parameter for overall and relapse-free survival (relative-risk (RR) of death, 4.6 (95% CI: 2.0-11.0; p < 0.001), RR of relapse, 7.2 (95% CI: 2.8-18.3; p < 0.001)). In this study, we demonstrate that TNFRSF10D DNA-methylation analysis of a small tissue-punch from archival FFPE melanoma tissue is a promising approach to provide prognostic information in patients with melanoma.

A reverse genetics approach to study feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a lethal immunopathological disease caused by feline coronaviruses (FCoVs). Here, we describe a reverse genetics approach to study FIP by assessing the pathogenicity of recombinant type I and type II and chimeric type I/type II FCoVs. All recombinant FCoVs established productive infection in cats, and recombinant type II FCoV (strain 79-1146) induced FIP. Virus sequence analyses from FIP-diseased cats revealed that the 3c gene stop codon of strain 79-1146 has changed to restore a full-length open reading frame (ORF).

Intraoperative Carotid Artery Injuries. Review of the literature, analysis of the material of one centre.

<b>Introduction:</b> Injuries of the carotid artery constitute a rare group of injuries. This study presents results of the treatment of 44 patients with iatrogenic carotid artery injuries for over 20 years. The patients were treated by the team of doctors of the Department of Vascular, General and Transplant Surgery in Wroclaw in the years 1997-2017 (Head of the Department Prof. Klemens Skóra, MD, and Prof. Piotr Szyber, MD, PhD - material used with permission). </br></br> <b>Aim:</b> Aims of the analysis are: to estimate the frequency of different forms of iatrogenic injuries to the common and internal carotid artery, to evaluate the results of treatment, to assess the most effective surgical method depending on the type of injury, and develop an effective preoperative, intraoperative and postoperative regimen.</br></br> <b>Discussion and results:</b> The frequency of various carotid artery injuries (blunt, acute, traffic) was constant between years, but the number of iatrogenic injuries definitely increased over time. The prognosis for patients with carotid artery injury, especially when combined with multi-organ trauma, is the gravest. Significantly better treatment results were achieved with both acute and iatrogenic injuries. This is mainly due to easier and quicker diagnosis and better conditions for assisting patients.</br></br> <b>Conclusions:</b> In iatrogenic injuries, a well-designed surgical scheme, i.e. primarily the administration of UTH and placing a temporary flow drain by the first operating team, reduces the risk of neurological complications.

Chimeric feline coronaviruses that encode type II spike protein on type I genetic background display accelerated viral growth and altered receptor usage.

Persistent infection of domestic cats with feline coronaviruses (FCoVs) can lead to a highly lethal, immunopathological disease termed feline infectious peritonitis (FIP). Interestingly, there are two serotypes, type I and type II FCoVs, that can cause both persistent infection and FIP, even though their main determinant of host cell tropism, the spike (S) protein, is of different phylogeny and displays limited sequence identity. In cell culture, however, there are apparent differences. Type II FCoVs can be propagated to high titers by employing feline aminopeptidase N (fAPN) as a cellular receptor, whereas the propagation of type I FCoVs is usually difficult, and the involvement of fAPN as a receptor is controversial. In this study we have analyzed the phenotypes of recombinant FCoVs that are based on the genetic background of type I FCoV strain Black but encode the type II FCoV strain 79-1146 S protein. Our data demonstrate that recombinant FCoVs expressing a type II FCoV S protein acquire the ability to efficiently use fAPN for host cell entry and corroborate the notion that type I FCoVs use another main host cell receptor. We also observed that recombinant FCoVs display a large-plaque phenotype and, unexpectedly, accelerated growth kinetics indistinguishable from that of type II FCoV strain 79-1146. Thus, the main phenotypic differences for type I and type II FCoVs in cell culture, namely, the growth kinetics and the efficient usage of fAPN as a cellular receptor, can be attributed solely to the FCoV S protein.

Multidisciplinary investigation of an unusual apparent homicide/suicide.

The investigation of deaths of individuals whose bodies are decomposed, mummified, or skeletonized is particularly difficult for medical examiners and medicolegal death investigators. Determination of the cause and manner of death in such cases frequently requires consultation with experts in a variety of disciplines in the forensic sciences and necessitates correlation of the autopsy results, scene investigation, medical and social history of the deceased, and laboratory studies. The authors report an unusual case of an apparent homicide/suicide involving 2 individuals and a canine that went undetected for almost 4 years. Determination of the cause and manner of death in these cases involved a multidisciplinary, intercontinental investigation and necessitated the performance of toxicologic studies on specimens not commonly analyzed. These cases illustrate the importance of the multidisciplinary approach to medicolegal death investigations and the necessity of considering nontraditional sources of potential information and specimens for laboratory analysis in selected cases.

Parasailing fatalities in southwest Florida.

Parasailing is a recreational sport that is generally considered to be of little risk to the participants. Typically, the passenger launches from a motorboat with a specially designed winch that pulls him or her back to the boat at the end of the ride. The sport is not regulated at the federal, state, or county level. There have been few reports of injuries to parasailors. Additionally, there have been only 2 fatalities reported to the United States Coast Guard in a 10-year review. We report the details of these 2 deaths, those of a mother and daughter riding in a tandem parasail, which occurred on Fort Myers Beach in 2001, as well as an additional case of a parasailing fatality that occurred in southwest Florida in 1999. These cases illustrate the injuries seen in such fatalities and the hazards posed by adverse weather conditions and faulty equipment, as well as the impairment of passenger judgment by drugs and/or alcohol.

Investigative and autopsy findings in sport aircraft-related deaths in southwest Florida.

Aircraft designated for sport or recreational use only, including ultralights, experimental aircraft and light-sport aircraft, have become increasingly popular. Because of their relative safety and the rarity of fatalities resulting from crashes of these aircraft, the forensic literature contains little information concerning the pathologic findings in such deaths. We report 9 deaths resulting from 6 sport aircraft crashes in southwest Florida, 6 pilots and 3 passengers. The vehicles involved 3 experimental aircraft, 1 ultralight and 2 "ultralight-like" aircraft. The patterns of injuries included trauma predominantly to the chest (3 cases), abdomen (1) or head (1), as well as multiple blunt force injuries involving the chest and abdomen (1) or the head and torso (3). Extremity fractures were found in only 2 cases, whereas injuries to the symphysis pubis were found in six. No "control-type" injuries were identified. These cases illustrate the varied pathologies associated with deaths due to crashes of sport aircraft and reveal the lack of uniformity associated with the investigations of such deaths.

Case report of suicide by inhalation of nitrogen gas.

Nitrogen is an inert gas that is a normal constituent of the air that we breathe. It is a suffocating gas that does not support life and that can be a cause of death by the displacement of oxygen in the atmosphere. The majority of deaths associated with nitrogen have occurred in the setting of scuba diving. Although other suffocating gases have been used as a means of committing suicide, the literature contains little information about the use of nitrogen as a suicidal agent. A case of a 50-year-old man who committed suicide using a homemade suicide device and nitrogen gas is presented.

Patterns of injury in a fatal BASE jumping accident.

Skydiving is a popular and relatively safe sport. The patterns of injury and mechanisms of death in the rare fatalities resulting from skydiving accidents have been well-documented. In contrast, BASE jumping, that is, jumping from a fixed object using a parachute, is a more dangerous and unregulated sport practiced by few individuals. There are no reports in forensic literature documenting the injuries found in deaths occurring in the practice of BASE jumping. We report the case of the death of an experienced BASE jumper who died after jumping from the antenna of a radio broadcast tower in southwest Florida to illustrate the unique hazards inherent in this sport.

Reverse Genetics for Type I Feline Coronavirus Field Isolate To Study the Molecular Pathogenesis of Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP), one of the most important lethal infections of cats, is caused by feline infectious peritonitis virus (FIPV), the high-virulence biotype of feline coronaviruses (FCoVs). FIPVs are suggested to emerge from feline enteric coronaviruses (FECVs) by acquiring mutations in specific genes in the course of persistent infections. Although numerous studies identified mutations predicted to be responsible for the FECV-FIPV biotype switch, the presumed roles of specific genetic changes in FIP pathogenesis have not been confirmed experimentally. Reverse genetics systems established previously for serotype I and the less common serotype II FCoVs were based on cell culture-adapted FIPV strains which, however, were shown to be unsuitable for FIP pathogenesis studies in vivo To date, systems to produce and manipulate recombinant serotype I field viruses have not been developed, mainly because these viruses cannot be grown in vitro Here, we report the first reverse genetics system based on a serotype I FECV field isolate that is suitable to produce high-titer stocks of recombinant FECVs. We demonstrate that these recombinant viruses cause productive persistent infections in cats that are similar to what is observed in natural infections. The system provides an excellent tool for studying FCoVs that do not grow in standard cell culture systems and will greatly facilitate studies into the molecular pathogenesis of FIP. Importantly, the system could also be adapted for studies of other RNA viruses with large genomes whose production and characterization in vivo are currently hampered by the lack of in vitro propagation systems.IMPORTANCE The availability of recombinant serotype I FCoV field isolates that are amenable to genetic manipulation is key to studying the molecular pathogenesis of FIP, especially since previous studies using cell culture-adapted FIPVs had proven unsuccessful. To our knowledge, we report the first serotype I FECV field isolate-based reverse genetics system that allows the production of high-titer recombinant virus stocks that can be used for subsequent in vivo studies in cats. The system represents a milestone in FCoV research. It provides an essential tool for studying the molecular pathogenesis of FIP and, more specifically, the functions of specific gene products in causing a fundamentally different progression of disease following acquisition of specific mutations. The system developed in this study will also be useful for studying other coronaviruses or more distantly related RNA viruses with large genomes for which suitable in vitro culture systems are not available.

Accidental boric acid poisoning following the ingestion of household pesticide.

Borate-containing compounds were formerly used as topical antiseptics and were components of many medicinal preparations including skin powders and ointments used for the treatment of burns and diaper rash. These compounds were also used as irrigants for body cavities, including the pleural, vaginal, and rectal cavities. These applications were subsequently discontinued by the medical community when the toxicity and potential lethality of borates were recognized. Although documented cases of borate poisoning are now rare, the chemical is still an active component commonly used in high concentrations in household disinfectants/cleaners, pesticides, and wood preservatives. While the majority of documented borate-related deaths have occurred in infants, the toddler population is currently at risk due to possible exposure to these household products. We present the case of an 18-month-old child who died following the accidental ingestion of a boric acid-containing, commercially available roach pesticide product.

Independence day explosion on lovers key.

The display of fireworks is a popular holiday celebration in the United States. Because injuries due to recreational fireworks-related explosions among private consumers are relatively common, the sale of fireworks is regulated by the federal government and is also limited by state and local laws. In contrast, because fireworks display companies are under tight safety regulations, explosions in the professional pyrotechnics industry are uncommon occurrences, and the literature contains rare reports of injuries and fatalities resulting from such explosions. We report the 2003 Fourth of July commercial fireworks explosion on Lovers Key in southwest Florida that resulted in five fatalities. Events occurring during the investigation of the scene of this explosion illustrate the unique considerations and hazards for medicolegal death investigators, law enforcement and other investigative agencies. Additionally, this case demonstrates unusual aspects of the postmortem examinations performed on victims of fireworks-related incidents.

Paths to destruction: the lives and crimes of two serial killers.

Although research into the phenomenon of serial murder has revealed that serial killers frequently do not fit the initially described paradigm in terms of their physical and psychological profiles, backgrounds, and motives to kill, the media continues to sensationalize the figures of such killers and the investigators who attempt to analyze them on the basis of aspects of their crimes. Although the so-called "typical" profile of the serial murderer has proven accurate in some instances, in many other cases the demographics and behaviors of these killers have deviated widely from the generalized assumptions. This report details two unusual cases in which five and eight murders were committed in upstate New York. The lives and crimes of these offenders illustrate the wide spectrum of variations in the backgrounds, demographics, motivations, and actions witnessed among serial murderers, and highlight the limitations and dangers of profiling based on generalities.

Sudden cardiac death due to giant cell inflammatory processes.

Granulomatous inflammation of the myocardium may occur in a number of systemic disease processes including those with infectious etiologies such as fungal, mycobacterial and parasitic infections, as well as hypersensitivity reactions, and rarely autoimmune disorders. In many of these disorders, giant cells are components of the inflammatory infiltrate. Systemic granulomatous processes of unknown pathogenesis, most notably sarcoidosis, may also be associated with involvement of the myocardium. Occasionally, these disorders are associated with sudden death due to pathologic involvement of the heart. In contrast, giant cell myocarditis, also known as idiopathic myocarditis, a rare, frequently fulminant and fatal disorder of unknown etiology, is isolated to the heart and lacks systemic involvement. This disorder is most commonly diagnosed at autopsy. We present two cases in which sudden death resulted from a giant cell inflammatory process affecting the myocardium. Both individuals lacked antemortem diagnoses and collapsed at their respective places of employment. These cases compare and contrast the clinical and pathologic issues involved in the differential diagnoses of the subgroup of sudden cardiac deaths resulting from giant cell inflammatory processes that affect the myocardium, as well as the value of histologic examination and immunohistochemical studies.

Alligator attacks in southwest Florida.

The American alligator inhabits bodies of fresh water in Florida and other southeastern states. Although attacks on pets are frequent, alligator attacks on humans are relatively rare because of the animal's natural fear of man. Because of the rarity of attacks on humans, the pathologic findings and pathophysiology of death in such cases have not been well characterized in the literature. We report three cases of fatal alligator attacks that occurred in southwest Florida, each with different pathologic findings and mechanisms of death. Although the cause of death in each case was attributed to the alligator attack, the mechanisms of death differed and included exsanguination because of amputation of an extremity, overwhelming sepsis, and drowning. These cases illustrate the varied pathophysiologies associated with deaths due to alligator attacks on humans and the features that distinguish alligator bites from those of other aquatic predators.

High prevalence of side population in human cancer cell lines.

Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems.

The stages of change questionnaire as a predictor of trauma patients most likely to decrease alcohol use.

BACKGROUND: Research has shown that negative trauma-related consequences of drinking can predict readiness to change drinking behavior. These findings are confined to patients with positive blood alcohol levels at admission. The current study extends such findings by examining whether stage of readiness to change among all adult trauma activations admitted to the emergency department for 24 hours or more can be used to predict a change in behavior at followup. STUDY DESIGN: Patients 18 years of age or older admitted to a Level II trauma center between December 1, 2001, and January 31, 2003, with a trauma activation were eligible to participate. Enrolled patients were screened for alcohol use and readiness to change with the Alcohol Use Disorders Identification Test (AUDIT) and Short Form Stages of Change (SFSC), respectively, within 2 days of discharge. Blood alcohol level (BAL) was obtained for all study patients. Six to 18 months after discharge, patients were followed up with the AUDIT. The ability of the SFSC to predict change in alcohol behavior at followup was analyzed by multiple regression. RESULTS: Of 253 eligible patients, 146 patients were enrolled and had BAL taken. Most were men (64%) and 36% were women. Mean positive BAL (n = 57) was 186 mg/dL (range 10 to 537 mg/dL). Of these, 23% (13 of 57) met AUDIT criteria for harmful drinking and 47% (27 of 57) met criteria for dependent drinking. Even among those with undetectable BAL (n = 89), harmful or dependent drinking was identified by the AUDIT for 11% (10 of 89). A readiness to change was common among those reporting harmful or dependent drinking (26%) at baseline. The SFSC independently and significantly predicted change in drinking behavior among the 72 patients with followup (p = 0.05). Patients indicating greater readiness to change at baseline were those more likely to decrease their consumption pattern at followup. CONCLUSIONS: Findings suggest that administering the brief SFSC questionnaire to all trauma patients and providing assistance to those demonstrating a willingness to change may prove to be an effective strategy for reducing problem alcohol use.