Discovery of potent and selective HER2 inhibitors with efficacy against HER2 exon 20 insertion-driven tumors, which preserve wild-type EGFR signaling.

Oncogenic alterations in human epidermal growth factor receptor 2 (HER2) occur in approximately 2% of patients with non-small cell lung cancer and predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. Most clinical-grade tyrosine kinase inhibitors are limited by either insufficient selectivity against wild-type (WT) epidermal growth factor receptor (EGFR), which is a major cause of dose-limiting toxicity or by potency against HER2 exon 20 mutant variants. Here we report the discovery of covalent tyrosine kinase inhibitors that potently inhibit HER2 exon 20 mutants while sparing WT EGFR, which reduce tumor cell survival and proliferation in vitro and result in regressions in preclinical xenograft models of HER2 exon 20 mutant non-small cell lung cancer, concomitant with inhibition of downstream HER2 signaling. Our results suggest that HER2 exon 20 insertion-driven tumors can be effectively treated by a potent and highly selective HER2 inhibitor while sparing WT EGFR, paving the way for clinical translation.

Dental evidence of human remains of a Slavic soldier population deported in concentration camps during World War II.

Aims: The aim of this paper was to analyse post-mortem dental records of a collection of 93 skeletal remains exhumed from the ossuary of Bari Municipal Cemetery, Apulia, Italy. These skeletal remains belonged to Slavic soldiers deported in 1941 during World War II in two Italian concentration camps and who died in 1946-1947. Methods: A total number of 1949 teeth were analysed according to the American Board of Forensic Odontology and the WHO methods. The majority of the victims were men (95%) between the age of 18 and 62. Results: The results showed the dental health situation of a population of soldiers of World War II, in particular the high rate of caries (35%), periodontal disease (61%), and dental wear (65%) according to the high stress level of the soldiers during the armed conflict and the subsequent deportation. Conclusions: This is the first study reported in literature that analyses the effects of war and deportation on soldiers' pathological conditions of the oral cavity. This analysis also confirmed the usefulness of teeth for anthropological and forensic research thanks to their high resistance and preservation even after post-mortem modifications and different environmental factors.

Torkildsen's Ventriculocisternostomy First Applications: The Anthropological Evidence of a Young Slavic Soldier Who Died in the Torre Tresca Concentration Camp (Bari, Italy) in 1946.

Human skeletal remains are considered as real biological archives of each subject's life. Generally, traumas, wounds, surgical interventions, and many human pathologies suffered in life leave identifiable marks on the skeleton, and their correct interpretation is possible only through a meticulous anthropological investigation of skeletal remains. The study here presented concerns the analysis of a young Slavic soldier's skeleton who died, after his imprisonment, in the concentration camp of Torre Tresca (Bari, Italy), during the Second World War (1946). In particular, the skull exhibited signs of surgical activity on the posterior cranial fossa and the parieto-occipital bones. They could be attributed to surgical procedures performed at different times, showing various degrees of bone edge remodeling. Overall, it was possible to correlate the surgical outcomes highlighted on the skull to the Torkildsen's ventriculocisternostomy (VCS), the first clinically successful shunt for cerebrospinal fluid (CSF) diversion in hydrocephalus, which gained widespread use in the 1940s. For this reason, the skeleton we examined represents a rare, precious, and historical testimony of an emerging and revolutionary neurosurgical technique, which differed from other operations for treating hydrocephalus before the Second World War and was internationally recognized as an efficient procedure before the introduction of extracranial shunts.

Sex determination through the evaluation of sella turcica measurements using head CT scan.

The estimation of age and sex is mandatory in forensic identification of unknown skeletal remains and it could be reached analyzing different bones' features. The morphometric evaluation of Sella turcica is less described in literature. Many studies have been published in this field for age estimation but none of them based on head CT scan measurement. Previous studies were carried out for age estimation on different populations, but any study has been carried out on the Italian population. Furthermore, no studies were carried out with CT scan technology for the correlation between sex and Sella turcica. Thanks to CT scan technology it has been possible to measure sella turcica's dimensions in an easier way than previous methods, to detect more accurate parameters and even introduce a new one. In this study, the sample was composed of 115 adults, 53 males, and 62 females. Five different measurements of the sellae turcicae were taken. The purpose of this study was to find new elements for sex assessment with the evaluation of sella turcica measurements, improving previous studies, analyzing an Italian European sample. In this study, results of the statistical analysis showed correlation between sex and sella turcica's parameter "Length of Sella Turcica" for which grater values are related to male gender. In conclusion, the Authors consider this unexpected result useful for sex determination in association with other parameters.

Dental age estimation: Demirjian's versus the other methods in different populations. A literature review.

Age estimation is an important issue in forensic anthropology and odontology, since it is applied to both the living and the dead. Studies have shown that assessing tooth development using radiological methods is a reliable way to estimate age, especially in children. Demirjian's method is the most widely used estimation method for dental age evaluation. Over the years, many methods have been developed in order to asses which one predicts more accurately the age. In the literature, many populations were examined with these different methods and discordant results are described. This review aims to evaluate the studies in which Demirjian's method was compared with Willems', Cameriere's, Nolla's, Smith's, Haavikko's and Chaillet's methods in order to estimate the most accurate dental age estimation method in different populations. Using the methodology described in the Preferred Reporting Items for Systematic Reviews and Meta-Analysis statement, the authors conducted a review of existing literature using three databases: PubMed, Google Scholar and Google Scopus.

Soft-tissue thickness values using cone beam computed tomography: A literature review.

When human remains are found, with no evidence of identity, facial approximation can be a useful technique to employ. The reconstruction of the ante-mortem appearance can reproduce the likely features of the face, starting from the skull, based on the overlying soft-tissue thickness. Over the years, many techniques have been developed to achieve soft-tissue thickness measurements, one of which is based on the use of cone beam computed tomography. This study aimed to review the status of this technique and to evaluate heterogeneity among studies undertaken in this field, with particular regard to determination of landmarks, sex and body mass index.

DNA extraction from sternum bone for identification of a saponified body: use of a modified protocol.

This paper discusses our approach and results obtained when attempting to identify a saponified human body recovered from the sea, without arms and legs. Bones, especially the long ones, are the only sources of DNA available in several cases involving unidentified bodies in advanced state of putrefaction. In this case, since the body was found without limbs, attempts were made to extract DNA from the sternum bone. The DNA was extracted using a modified version of the NucleoSpin® DNA Trace Kit (Macherey Nagel™) protocol and an STR analysis was performed. Thanks to this modified protocol a complete DNA profile was obtained from the sternum bone, while only partial results were obtained from blood and teeth. The DNA profile obtained from the sternum was compared with the DNA of the putative son searching for a genetic match. Five incompatibilities were detected so it was possible to exclude the kinship. In conclusion this could be a useful technique in personal identification through DNA analysis in case of poor quality and quantity of bone.

SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism.

SLC25A32 is a member of the solute carrier 25 family of mitochondrial transporters. SLC25A32 transports tetrahydrofolate (THF) as well as FAD into mitochondria and regulates mitochondrial one-carbon metabolism and redox balance. While it is known that cancer cells require one-carbon and FAD-dependent mitochondrial metabolism to sustain cell proliferation, the role of SLC25A32 in cancer cell growth remains unexplored. Our results indicate that the SLC25A32 gene is highly amplified in different tumors and that amplification correlates with increased mRNA expression and reduced patients´ survival. siRNA-mediated knock-down and CRISPR-mediated knock-out of SLC25A32 in cancer cells of different origins, resulted in the identification of cell lines sensitive and resistant to SLC25A32 inhibition. Mechanistically, tracing of deuterated serine revealed that SLC25A32 knock-down does not affect the mitochondrial/cytosolic folate flux as measured by Liquid Chromatography coupled Mass Spectrometry (LC-MS). Instead, SLC25A32 inhibition results in a respiratory chain dysfunction at the FAD-dependent complex II enzyme, induction of Reactive Oxygen Species (ROS) and depletion of reduced glutathione (GSH), which impairs cancer cell proliferation. Moreover, buthionine sulfoximine (BSO) treatment further sensitizes cells to ROS-mediated inhibition of cell proliferation upon SLC25A32 knock-down. Treatment of cells with the FAD precursor riboflavin and with GSH rescues cancer cell proliferation upon SLC25A32 down-regulation. Our results indicate that the reduction of mitochondrial FAD concentrations by targeting SLC25A32 has potential clinical applications as a single agent or in combination with approved cancer drugs that lead to increased oxidative stress and reduced tumor growth.

Management of severe complications following penile surgery for erectile dysfunction and Peyronie disease: Three case reports.

RATIONALE: Erectile dysfunction (ED) and Peyronie's disease (PD) are conditions commonly observed in andrology. Despite the surgical refinement and the technical improvement in this field, even in expert hands, detrimental consequences have been reported and it can be related to patient's comorbidities or misconduct in the postoperative period. In this article we report anecdotal cases of severe complications following penile surgery for ED and PD in high volume centers, describe the strategies adopted to treat it and discuss the options that would have helped preventing these events. PATIENTS' CONCERNS: The first case describes a patient with history of ED and PD causing penile shortening and a slight dorsal deviation of penile shaft. In the second case it is described a corporeal necrosis and urethral fistula following inflatable penile prosthesis implant. In the last case it is described the migration of reservoir into the abdomen after inflatable penile prosthesis implantation post-radical prostatectomy. DIAGNOSIS: All 3 patients were investigated with a penile doppler ultrasound with PGE1 intracorporeal injection for ED and PD diagnosis. An abdominal computed tomography scan and magnetic resonance imaging were ordered for patient of case three. INTERVENTIONS: The patients underwent different combined procedures depending on the case and including: glansectomy, penile prosthesis implantation associated with a penile elongation with double dorsal-ventral patch graft ("sliding technique"), penile urethroplasty with buccal mucosa graft, and laparotomy for reservoir removal. OUTCOMES: No further serious complications were reported after the procedures described. LESSONS: Penile surgery in patients with concomitant PD and systemic comorbidities can be at high risk of complications. As shown in this series there are possible dramatic evolution of these complications that may cause irreversible consequences to the patient. For this reason, a dedicated surgical and nursing team is necessary to reduce the chances that it happens. When this event occurs, a team trained in their management can improve the patient outcome.

A Comparison of 3 Established Skeletal Age Estimation Methods in an African Group From Benin and an Italian Group From Southern Italy.

Age estimation is a crucial matter in several forensic and legal instances. The literature recommends carrying out hand and wrist radiograph to assess skeletal age. Much research has been conducted to examine the application of various methods in different categories. Our study aims to evaluate the reliability of Greulich and Pyle, Tanner-Whitehouse 2, and FELS methods in Beninese and Italian samples, with a total of 204 subjects, 102 (51 were male and 51 were female) from Benin and 102 (51 were male and 51 were female) from Italy, aged between 4 and 19 years. Hand-wrist radiographs were carried out at Saint Luc Hospital in Cotonou for the African sample and in 2 hospitals in Southern Italy for Italian sample. Skeletal maturity was obtained, applying the 3 methodologies. The arithmetic mean of the 3 ages obtained was calculated, as well as the possible difference between the known chronological age and the estimated mean skeletal age. Data were analyzed statistically using the χ test, Student t test, and Bland-Altman plots. There are no significant statistical differences between real age and skeletal age calculated using the Greulich and Pyle and FELS methods. Comparison between Tanner-Whitehouse 2 and the average of the methods used results in statistically significant, but clinically insignificant, differences.

Facial approximation: Soft tissue thickness values for Caucasian males using cone beam computer tomography.

The aim of this study was to obtain soft tissue thickness (STTs) of Caucasian males starting from 12 bone landmarks, to allow facial reconstruction and assign an identity to skeletal remains. When a skeleton is found without any circumstantial evidence of identity, through anthropological investigations it is possible to estimate age, sex, and race studying specific characteristics of bones. This first step of identification process helps investigators to narrow down the identity suppositions, but not always forensic analysis guarantees a final positive identification. Moreover, it is possible to rely on facial reconstruction trying to reproduce the likely features of the face from the skull. The obtained images could be used as a support in recognition by relatives who could provide photos and other data to carry out a comparative analysis. Furthermore, identity can be confirmed by conventional identification methods such as DNA profiling, odontological identification, and radiological comparison. Forensic facial reconstruction (FFR) is based on the bone surface that conditions the facial physiognomy. Analyzing cone beam CT (CBCT) scans of 30 Caucasian males, average STTs values were obtained. CBCT, compared with the conventional spiral CT, has much lower radiation doses, lower costs, high spatial resolution and focuses on head and neck. The obtained results were compared with existing literature and databases and were analyzed by descriptive statistics. Two researchers repeated measurements in different times to assess the reliability of the method.

Photogrammetric 3D skull/photo superimposition: A pilot study.

The identification of bodies through the examination of skeletal remains holds a prominent place in the field of forensic investigations. Technological advancements in 3D facial acquisition techniques have led to the proposal of a new body identification technique that involves a combination of craniofacial superimposition and photogrammetry. The aim of this study was to test the method by superimposing various computerized 3D images of skulls onto various photographs of missing people taken while they were still alive in cases when there was a suspicion that the skulls in question belonged to them. The technique is divided into four phases: preparatory phase, 3d acquisition phase, superimposition phase, and metric image analysis 3d. The actual superimposition of the images was carried out in the fourth step. and was done so by comparing the skull images with the selected photos. Using a specific software, the two images (i.e. the 3D avatar and the photo of the missing person) were superimposed. Cross-comparisons of 5 skulls discovered in a mass grave, and of 2 skulls retrieved in the crawlspace of a house were performed. The morphologyc phase reveals a full overlap between skulls and photos of disappeared persons. Metric phase reveals that correlation coefficients of this values, higher than 0.998-0,997 allow to confirm identification hypothesis.

Lysine acetylation in mitochondria: From inventory to function.

Cellular signaling pathways are regulated in a highly dynamic fashion in order to quickly adapt to distinct environmental conditions. Acetylation of lysine residues represents a central process that orchestrates cellular metabolism and signaling. In mitochondria, acetylation seems to be the most prevalent post-translational modification, presumably linked to the compartmentation and high turnover of acetyl-CoA in this organelle. Similarly, the elevated pH and the higher concentration of metabolites in mitochondria seem to favor non-enzymatic lysine modifications, as well as other acylations. Hence, elucidating the mechanisms for metabolic control of protein acetylation is crucial for our understanding of cellular processes. Recent advances in mass spectrometry-based proteomics have considerably increased our knowledge of the regulatory scope of acetylation. Here, we review the current knowledge and functional impact of mitochondrial protein acetylation across species. We first cover the experimental approaches to identify and analyze lysine acetylation on a global scale, we then explore both commonalities and specific differences of plant and animal acetylomes and the evolutionary conservation of protein acetylation, as well as its particular impact on metabolism and diseases. Important future directions and technical challenges are discussed, and it is pointed out that the transfer of knowledge between species and diseases, both in technology and biology, is of particular importance for further advancements in this field.

Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer.

BACKGROUND: The antibody cetuximab, targeting epidermal growth factor receptor (EGFR), is used to treat metastatic colorectal cancer (mCRC). Clinical trials suggest reduced benefit from the combination of cetuximab with oxaliplatin. The aim of this study was to investigate potential negative interactions between cetuximab and oxaliplatin. METHODS: Thiazolyl blue tetrazolium bromide (MTT) assay and Calcusyn software were used to characterize drug interactions. Reactive oxygen species (ROS) were measured by flow cytometry and real-time polymerase chain reaction oxidative stress arrays identified genes regulating ROS production. Chromatin immunoprecipitation (ChIP) measured signal transducer and activator of transcription 1 (STAT-1) binding to dual oxidase 2 (DUOX2) promoter. SW48, DLD-1 KRAS wild-type cell lines and DLD-1 xenograft models exposed to cetuximab, oxaliplatin, or oxaliplatin + cetuximab (control [saline]; n = 3 mice per treatment group) were used. Statistical tests were two-sided. RESULTS: Cetuximab and oxaliplatin exhibited antagonistic effects on cellular proliferation and apoptosis (caspase 3/7 activity reduced by 1.4-fold, 95% confidence interval [CI] = 0.78 to 2.11, P = .003) as opposed to synergistic effects observed with the irinotecan metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38). Although both oxaliplatin and SN-38 produced ROS, only oxaliplatin-mediated apoptosis was ROS dependent. Production of ROS by oxaliplatin was secondary to STAT1-mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.41, P < .001). Inhibition of DUOX2 induction and p38 activation by cetuximab reduced oxaliplatin cytotoxicity. CONCLUSIONS: Inhibition of STAT1 and DUOX2-mediated ROS generation by cetuximab impairs p38-dependent apoptosis by oxaliplatin in preclinical models and may contribute to reduced efficacy in clinical settings. Understanding the rationale for unexpected trial results will inform improved rationales for combining EGFR inhibitors with chemotherapeutic agents in future therapeutic use.

Validity Comparison of Three Dental Methods for Age Estimation Based on Tooth Root Translucency.

Age estimation of nonliving subjects plays a very important role in identifying human remains. When adult remains are in question, this process becomes even more complex. Among the numerous dental methods that exist for adults aging, the evaluation of root translucency is one of the most useful. This study studies the correlation between age and root translucency by investigating monoradicular teeth affected by pathological processes using the equations proposed by Lamendin, Prince & Ubelaker and from Singhal et al. The average age values calculated according to the three methods were compared with the values of known age. Age estimation using the Student's t-test resulted as more accurate in the methods by Singhal et al. and by Prince & Ubelaker, with respect to that of Lamendin. Age estimation based solely on the percentage of radicular dentinal translucency has been found to be more accurate when diseased teeth are involved.

Assessment of legal adult age of 18 by measurement of open apices of the third molars: Study on the Albanian sample.

The third molar tooth is one of the few anatomical structures in development available for estimating the age of individuals in the late adolescence. This study tests the accuracy of Cameriere's cut-off value of the third molar index (I3M) in assessing legal adult age of 18 years in an Albanian sample. For this purpose, a sample of orthopantomograms (OPTs) of 286 living subjects (152 female and 134 male) aged between 15 and 22 years was analyzed. Intra-rater and inter-raters agreement of I3M were 0.998 and 0.998, respectively and Cohen Kappa for intra-rater and inter-rater agreement in decision on adult or minor was 1.0 and 1.0, respectively. Age distribution gradually decreases as I3M increases in both males and females. The mean age of females is higher than that of males when I3M is between 0.04 and 0.08. Sensitivity test for males was 94.1%, with a 95% confidence interval (95% CI) 85.6-98.4%, and specificity was 90.9% (95%CI 81.3-96.6%). The proportion of correctly classified individuals was 92.5%, with a 95%CI of (86.7%, 96.4%). For females, the sensitivity test was 75.4%, with a 95%CI of (63.1%, 85.2%) and specificity was 96.6%, with a 95%CI of (90.3%, 99.3%). The proportion of correctly classified individuals was 87.5%, with a 95%CI of (81.2%, 92.3%). The results indicate that Cameriere's cut-off value of the third molar index (I3M=0.08) is useful in discriminating between Albanian adults and juveniles, and encourage us to test its suitability for determining the adult age in individuals from other populations.

A case of an adoptive girl with precocious puberty: the problem of age estimation.

Age estimation is one of the main tasks of forensic anthropology and odontology, both on the dead and the living. In living subjects, age estimation may be used to establish an individual's status as a minor in cases involving adoption, criminal responsibility, child pornography, and those seeking asylum, especially where adequate identification documents are lacking. The authors report a case about age assessment of a girl born in Mbujimayi (Democratic Republic of Congo) and later adopted in Italy. The birth certificate issued after finding the child in a state of abandonment (in December 2007), bore date of 12.12.2004, but this was in contrast with the year of birth - 2003 - stated on the certification available to the center that had provided accommodation to the girl in Africa. Her adoptive parents reported that the child had been diagnosed with precocious puberty and was thus under treatment. She weighed 32.5 kg and was 132.5 cm tall. Body mass index (BMI) corresponded to the range between 9.5 and 14 years of age. The assessment of maturity indicators (sexual characteristics) placed the child at the lower limits of Stage II of Tanner's classification (sparse growth of long, slightly darkened, downy straight pubic hair; elevation of the breast and nipple as a small mound with increased diameter of the areolae). The skeletal age was determined by taking X-rays of the hand and wrist using Fels, TW2 and Greulich and Pyle methods. Dental growth was assessed through orthopantomogram using Demirjian's technique. The methods applied were adjusted considering the studies on African population found in the literature, and a skeletal and dental age of 10 years was established. Afterwards, the wrist X-rays performed at the Children's Hospital of Bari, 7 months before our investigation, revealed a skeletal age of 7 years. This evidence showed that, despite the treatment the child had promptly initiated, early puberty had influenced the skeletal growth with an acceleration of about 2 years. Therefore, the age we detected (10 years) was the result of precocious puberty. Then, we backdated the girl's birth (by about 2 years, based on current studies), stating that she was about 8 at the time of our investigation. This case is significant both for the racial differences that need to be considered in age assessment, and for the importance of any pathology (growth disorders) that may affect physiological development and make the applied methods poorly reliable.

Age assessment using the Greulich and Pyle method on a heterogeneous sample of 300 Italian healthy and pathologic subjects.

The exponential growth of the illegal immigration phenomenon, the requests for asylum, and the increasing number of migrant settlements on Italian territory have strong repercussions on the legal system. One important aspect of this problem is age assessment. In the case of living individuals, identification may be focused on ascertaining their age, more directly if they can attest their age but are not registered (foreigners who claim to be minors or under 14 years of age). These have entered our country as illegal immigrants and have an interest in declaring a younger age (as a minor or under 14) rather than their true age because they can obtain the benefits specifically provided by Italian law for such categories. Since the most frequently used method in age assessment is the Greulich and Pyle Atlas, the objective of this study was to evaluate the consistency and accuracy of the method on a sample of Italian teenagers, in order to ascertain whether or not the Atlas is suitable for the purpose, especially in the critical threshold between 14 and 18 years of age. A total of 300 radiographs of the left wrist and hand of individuals between 10 and 20 years of age were obtained from the outpatient Pediatric Radiology Department of the Papa Giovanni XXIII Hospital in Bari. The results showed that the Greulich and Pyle Atlas, used on the Italian population, provide a good match with the chronological age, showing no statistically significant differences. The Greulich and Pyle Atlas is usable on the Italian population since there were no significant differences in skeletal age determination with this method as compared to the chronological age; however, one must allow for a possible standard deviation equal to more or less 13 months.

Extraction of DNA from bones in cases where expectations for success are low.

To resolve cases involving unidentified cadavers, the study of polymorphic DNA markers of old bones is an invaluable but often challenging tool used in forensic genetics. Some of the difficulties encountered involve the limited quantity of endogenous DNA, its subsequent degradation (a result of elapsed time, environmental conditions, and the microorganisms that develop during the postmortem phase), and the coextraction of substances that inhibit amplification reactions. For these reasons, it is necessary to direct research toward the development of new extraction techniques with the goal of obtaining adequate quantities of high-quality DNA.The aim of this study was to improve the collection of extracted DNA compared with the amount of DNA obtained with the NucleoSpin DNA Trace Kit (Macherey Nagel) protocol for the extraction of genomic DNA from human bones. A modified version of the standard protocol is presented.The modified method for the extraction of genomic DNA, followed by amplification reaction, allowed for identification of 4 cadavers and typification of 1 cadaver. The study carried out involved unidentified cadavers, or their remains, discovered after a long period from time of death.