Allele-specific CRISPR-Cas9 editing of dominant epidermolysis bullosa simplex in human epidermal stem cells.

Epidermolysis bullosa simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish the dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon 1. Through the CRISPR-Cas9 system, we perform a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

Rethinking the definition of 'less intensive' for venetoclax-combining regimens in acute myeloid leukaemia patients.

Invasive fungal infections (IFIs), mainly due to pulmonary aspergillosis, are considered a serious complication in acute leukaemia, with an unfavourable impact on patient. In this well-conducted retrospective study, Reynolds et al. suggest that the use of posaconazole prophylaxis in association with venetoclax plus hypomethylating agents or chemotherapy leads to a reduction of IFI incidence. Therapeutic drug monitoring of posaconazole levels is suggested, even if no correlation with IFI risk has been demonstrated. Commentary on: Reynolds et al. Invasive fungal infection following venetoclax and posaconazole co-administration. Br J Haematol 2023;203:593-598.

European Council of Legal Medicine (ECLM) on-site inspection forms for forensic pathology, anthropology, odontology, genetics, entomology and toxicology for forensic and medico-legal scene and corpse investigation: the Parma form.

Further to a previous publication by the European Council of Legal Medicine (ECLM) concerning on-site forensic and medico-legal scene and corpse investigation, this publication provides guidance for forensic medical specialists, pathologists and, where present, coroners' activity at a scene of death inspection and to harmonize the procedures for a correct search, detection, collection, sampling and storage of all elements which may be useful as evidence, and ensure documentation of all these steps. This ECLM's inspection form provides a checklist to be used on-site for the investigation of a corpse present at a crime or suspicious death scene. It permits the collection of all relevant data not only for the pathologist, but also for forensic anthropologists, odontologists, geneticists, entomologists and toxicologists, thus supporting a collaborative work approach. Detailed instructions for the completion of forms are provided.

The effect of the medico-legal evaluation on asylum seekers in the Metropolitan City of Milan, Italy: a pilot study.

In the present-day situation, the clinical forensic documentation of an asylum seeker's narrative and his or her examination, together with the physical and psychological findings, may have very important effects on the outcome of the request for political asylum. Since 2012, the Municipality of Milan, the University Institute of Legal Medicine, and other institutions have assembled a team with the task of examining vulnerable asylum seekers and preparing a medical report for the Territorial Commission for International Protection (Prefecture, Ministry of Interiors), who will assess the application. We compared medico-legal reports and outcomes of 57 cases which were evaluated by the Commission after having undergone a medico-legal evaluation through the Istanbul Protocol criteria and examined, in particular, which medico-legal variables seem associated to the outcome. The results show that forensic assessment seems to have a significant and interesting correlation with the final assessment given by the Commission. For example, the higher the level of consistency, according to the Istanbul Protocol, the more frequently protection is granted. These data show how important clinical forensic medicine can be in such scenarios and how the presence of clinical forensic experts should be encouraged in such evaluations, as has been recently enshrined in Italy in the guidelines of a Ministerial Decree of April 3rd, 2017 for the assistance and the rehabilitation as well as the treatment of psychiatric disorders in refugees and asylum seekers who have undergone torture, rape, and other severe forms of psychological, physical, or sexual violence.

Bloodstream infections in haematological cancer patients colonized by multidrug-resistant bacteria.

Infections by multidrug-resistant (MDR) bacteria are a worrisome phenomenon in hematological patients. Data on the incidence of MDR colonization and related bloodstream infections (BSIs) in haematological patients are scarce. A multicentric prospective observational study was planned in 18 haematological institutions during a 6-month period. All patients showing MDR rectal colonization as well as occurrence of BSI at admission were recorded. One-hundred forty-four patients with MDR colonization were observed (6.5% of 2226 admissions). Extended spectrum beta-lactamase (ESBL)-producing (ESBL-P) enterobacteria were observed in 64/144 patients, carbapenem-resistant (CR) Gram-negative bacteria in 85/144 and vancomycin-resistant enterococci (VREs) in 9/144. Overall, 37 MDR-colonized patients (25.7%) developed at least one BSI; 23 of them (62.2%, 16% of the whole series) developed BSI by the same pathogen (MDRrel BSI), with a rate of 15.6% (10/64) for ESBL-P enterobacteria, 14.1% (12/85) for CR Gram-negative bacteria and 11.1% (1/9) for VRE. In 20/23 cases, MDRrel BSI occurred during neutropenia. After a median follow-up of 80 days, 18 patients died (12.5%). The 3-month overall survival was significantly lower for patients colonized with CR Gram-negative bacteria (83.6%) and VRE (77.8%) in comparison with those colonized with ESBL-P enterobacteria (96.8%). CR-rel BSI and the presence of a urinary catheter were independent predictors of mortality. MDR rectal colonization occurs in 6.5% of haematological inpatients and predicts a 16% probability of MDRrel BSI, particularly during neutropenia, as well as a higher probability of unfavourable outcomes in CR-rel BSIs. Tailored empiric antibiotic treatment should be decided on the basis of colonization.

Macrophage activation syndrome in adult systemic lupus erythematosus: report of seven adult cases from a single Italian rheumatology center.

The aim was to describe the macrophage activation syndrome (MAS), a life-threatening syndrome characterized by excessive immune activation that can be triggered by conditions affecting immune homeostasis, in a cohort of adult Italian patients with systemic lupus erythematosus (SLE). This was a monocentric retrospective evaluation. The utility of the H-score, developed to estimate the individual risk of having reactive MAS in adult patients, was assessed. Among 511 patients with SLE, 7 cases (1.4%) of MAS (all females) were identified and their medical records reviewed. In all cases, MAS was simultaneous to the onset of SLE. All patients had fever, lymphadenopathy, hematological involvement, and high titer of anti-dsDNA antibodies. Workup for infections and malignancies was negative. In all cases, the H-score was higher than the cut-off suggested for the classification of reactive MAS. All cases required hospital admission, and 2 patients were admitted to the intensive care unit. Most patients were treated successfully with high doses of corticosteroids and with immunosuppressive drugs, whereas the full therapeutic regimen developed for primary hemophagocytic lymphohistiocytosis HLH was used only in one case. No death from MAS was observed. MAS is a rare and severe disorder that complicated the onset of SLE in our cohort. The H-score may be useful in the classification of these patients.

Skeletal idiopathic osteosclerosis helps to perform personal identification of unknown decedents: A novel contribution from anatomical variants through CT scan.

Personal identification consists of the comparison of ante-mortem information from a missing person with post-mortem data obtained from an unidentified corpse. Such procedure is based on the assessment of individualizing features which may help in providing a conclusive identification between ante-mortem and post-mortem material. Anatomical variants may provide important clues to correctly identify human remains. Areas of idiopathic osteosclerosis (IO), or dense bone islands (DBIs) characterized by radiopaque areas of dense, trabeculated, non-inflamed vital bone represent one of these, potentially individualizing, anatomical features. This study presents a case where the finding of DBI was crucial for a positive identification through CT-scan. A decomposed body was found in an apartment in June 2014 in advanced decomposition and no dental records were available to perform a comparison for positive identification. Genetic tests were not applicable because of the lack of relatives in a direct line. The analysis of the only ante-mortem documentation, a CT-scan to the deceased dating back to August 2009, showed the presence of three DBIs within the trabecular bone of the proximal portion of the right femur. The same bony district was removed from the corpse during the autopsy and analysed by CT-scan, which verified the presence of the same features. Forensic practitioners should therefore be aware of the great importance of anatomical bone variants, such as dense bone islands for identification purposes, and the importance of advanced radiological technique for addressing the individualizing potential of such variants. We propose that anatomical variants of the human skeleton should be considered as being "primary identification characteristics" similar to dental status, fingerprints and DNA.

Blood or spores? A cautionary note on interpreting cellular debris on human skeletal remains.

The identification of red blood cells on both skeletal human remains and decomposed corpses is of remarkable importance in forensic sciences, irrespective of its diagnostic value; their presence is often perplexing and difficult to interpret especially when in the context of decomposition and taphonomical variables. Some clinical research has focused on the morphological changes of red blood cells over time by scanning electron microscopy (SEM), but no research has investigated whether botanical structures can be confused for red blood cells. Since some literature has recently presumed the detection of erythrocyte-like cells on skeletal remains (even ancient) as surely erythrocytes, and most have never taken into consideration the chance of an origin different from blood, such as botanical, the present study aims at verifying the possibility of confusion between erythrocytes and botanical cells by applying SEM analysis and at highlighting the pitfalls in this particular issue through a test submitted to pathologists and natural scientists asked to discriminate between red blood cells and different vegetal structures (60 images obtained by SEM analysis). The results showed that although there are diagnostic features useful in identifying red blood cells from botanical structures, some spores resulted very similar to decaying red blood cells, which calls for attention and great caution when studying decomposed human remains.

Does cone beam CT actually ameliorate stab wound analysis in bone?

This study aims at verifying the potential of a recent radiological technology, cone beam CT (CBCT), for the reproduction of digital 3D models which may allow the user to verify the inner morphology of sharp force wounds within the bone tissue. Several sharp force wounds were produced by both single and double cutting edge weapons on cancellous and cortical bone, and then acquired by cone beam CT scan. The lesions were analysed by different software (a DICOM file viewer and reverse engineering software). Results verified the limited performances of such technology for lesions made on cortical bone, whereas on cancellous bone reliable models were obtained, and the precise morphology within the bone tissues was visible. On the basis of such results, a method for differential diagnosis between cutmarks by sharp tools with a single and two cutting edges can be proposed. On the other hand, the metrical computerised analysis of lesions highlights a clear increase of error range for measurements under 3 mm. Metric data taken by different operators shows a strong dispersion (% relative standard deviation). This pilot study shows that the use of CBCT technology can improve the investigation of morphological stab wounds on cancellous bone. Conversely metric analysis of the lesions as well as morphological analysis of wound dimension under 3 mm do not seem to be reliable.

Application of age estimation methods based on teeth eruption: how easy is Olze method to use?

The development of new methods for age estimation has become with time an urgent issue because of the increasing immigration, in order to estimate accurately the age of those subjects who lack valid identity documents. Methods of age estimation are divided in skeletal and dental ones, and among the latter, Olze's method is one of the most recent, since it was introduced in 2010 with the aim to identify the legal age of 18 and 21 years by evaluating the different stages of development of the periodontal ligament of the third molars with closed root apices. The present study aims at verifying the applicability of the method to the daily forensic practice, with special focus on the interobserver repeatability. Olze's method was applied by three different observers (two physicians and one dentist without a specific training in Olze's method) to 61 orthopantomograms from subjects of mixed ethnicity aged between 16 and 51 years. The analysis took into consideration the lower third molars. The results provided by the different observers were then compared in order to verify the interobserver error. Results showed that interobserver error varies between 43 and 57 % for the right lower third molar (M48) and between 23 and 49 % for the left lower third molar (M38). Chi-square test did not show significant differences according to the side of teeth and type of professional figure. The results prove that Olze's method is not easy to apply when used by not adequately trained personnel, because of an intrinsic interobserver error. Since it is however a crucial method in age determination, it should be used only by experienced observers after an intensive and specific training.

The persistence of ligature marks: towards a new protocol for victims of abuse and torture.

One of the most frequently observed lesions in clinical forensic practice concerns the patterned abrasion on skin due to constriction by various types of ligature. Detection of ligature marks and their patterns may be fundamental for reconstructing events and supporting testimony of an aggression, sexual abuse, or maltreatment. But very little actually exists in literature concerning their detectability and how long they last. This study aims at evaluating the time of persistence and detectability of skin signs left by different types of ligatures in living persons: on the arms of three volunteers, eight different ligatures were applied; 123 tests were performed, with time of contact ranging between 1 min and 2 h and 45 min. In addition, the persistence of the shape and pattern of the ligature was evaluated 15, 30, and 60 min after a 5- and 15-min compression. Polyvinyl siloxane, applied by a gun dispenser, was used to perform a cast of the skin mark. The results show that the pattern was less distinguishable with the decrease of time of contact, going from 75 % after 10 and 15 min of contact, to 45.8 % after 1 min. Above 15 min, the specific pattern was always recognizable. In addition, a progressive decrease of the detectability of the pattern with time, respectively, up to 12.5 and 37.5 % in 5- and 15-min tests was observed. This study provides useful results for the assessment of patterned injuries in forensic pathology and clinical forensic medicine, both on dead and living persons: in addition, the use of silicone casts seems to be a reliable and cheap method for easily recording and preserving the morphological profile of skin lesions.

The juvenile face as a suitable age indicator in child pornography cases: a pilot study on the reliability of automated and visual estimation approaches.

OBJECTIVE: In cases of suspected child pornography, the age of the victim represents a crucial factor for legal prosecution. The conventional methods for age estimation provide unreliable age estimates, particularly if teenage victims are concerned. In this pilot study, the potential of age estimation for screening purposes is explored for juvenile faces. In addition to a visual approach, an automated procedure is introduced, which has the ability to rapidly scan through large numbers of suspicious image data in order to trace juvenile faces. METHODS: Age estimations were performed by experts, non-experts and the Demonstrator of a developed software on frontal facial images of 50 females aged 10-19 years from Germany, Italy, and Lithuania. To test the accuracy, the mean absolute error (MAE) between the estimates and the real ages was calculated for each examiner and the Demonstrator. RESULTS: The Demonstrator achieved the lowest MAE (1.47 years) for the 50 test images. Decreased image quality had no significant impact on the performance and classification results. The experts delivered slightly less accurate MAE (1.63 years). Throughout the tested age range, both the manual and the automated approach led to reliable age estimates within the limits of natural biological variability. CONCLUSIONS: The visual analysis of the face produces reasonably accurate age estimates up to the age of 18 years, which is the legally relevant age threshold for victims in cases of pedo-pornography. This approach can be applied in conjunction with the conventional methods for a preliminary age estimation of juveniles depicted on images.

Multicentre surveillance study on feasibility, safety and efficacy of antifungal combination therapy for proven or probable invasive fungal diseases in haematological patients: the SEIFEM real-life combo study.

UNLABELLED: This multicentre observational study evaluated the feasibility, efficacy and toxicity of antifungal combination therapy (combo) as treatment of proven or probable invasive fungal diseases (IFDs) in patients with haematological malignancies. Between January 2005 and January 2010, 84 cases of IFDs (39 proven and 45 probable) treated with combo were collected in 20 Hematological Italian Centres, in patients who underwent chemotherapy or allogeneic haematopoietic stem cell transplantation for haematological diseases. Median age of patients was 34 years (range 1-73) and 37% had less than 18 years. Acute leukaemia was the most common underlying haematological disease (68/84; 81%). The phase of treatment was as follows: first induction in 21/84 (25%), consolidation phase in 18/84 (21%) and reinduction/salvage in 45/84 (54%). The main site of infection was lung with or without other sites. The principal fungal pathogens were as follows: Aspergillus sp. 68 cases (81%), Candida sp. six cases (8%), Zygomycetes four cases (5%) and Fusarium sp. four cases (5%). The most used combo was caspofungin+voriconazole 35/84 (42%), caspofungin + liposomal amphotericin B (L-AmB) 20/84 (24%) and L-AmB+voriconazole 15/84 (18%). The median duration of combo was 19 days (range 3-180). The overall response rate (ORR) was 73% (61/84 responders) without significant differences between the combo regimens. The most important factor that significantly influenced the response was granulocyte (PMN) recovery (P 0.009). Only one patient discontinued therapy (voriconazole-related neurotoxicity) and 22% experienced mild and reversible adverse events (hypokalaemia, ALT/AST increase and creatinine increase). The IFDs-attributable mortality was 17%. This study indicates that combo was both well tolerated and effective in haematological patients. The most used combo regimens were caspofungin + voriconazole (ORR 80%) and caspofungin + L-AmB (ORR 70%). The ORR was 73% and the mortality IFD related was 17%. PMN recovery during combo predicts a favourable outcome. CLINICAL TRIALS REGISTRATION: NCT00906633.

Synthesising 30 years of clinical experience and scientific insight on affective temperaments in psychiatric disorders: State of the art.

The concept of affective temperament has been extensively discussed throughout the history of psychopathology and represents a cornerstone in the study of mood disorders. This review aims to trace the evolution of the concept of affective temperaments (ATs) from Kraepelin's seminal work to the present day. In the 1980s, Akiskal redefined Kraepelin's concept of affective temperaments (ATs) by integrating the five recognized ATs into the broader framework of the soft bipolar spectrum. This conceptualization viewed ATs as non-pathological predispositions underlying psychiatric disorders, particularly mood disorders. Epidemiological and clinical studies have validated the existence of the five ATs. Furthermore, evidence suggests that ATs may serve as precursors to various psychiatric disorders and influence clinical dimensions such as disease course, psychopathology, and treatment adherence. Additionally, ATs appear to play a significant role in moderating phenomena such as suicide risk and stress coping. Incorporating an evaluation of temperamental bases of disorders into the multidimensional psychiatric diagnostic process could enhance treatment optimization and prognosis estimation.

Bioarchaeology aids the cultural understanding of six characters in search of their agency (Tarquinia, ninth-seventh century BC, central Italy).

Etruria contained one of the great early urban civilisations in the Italian peninsula during the first millennium BC, much studied from a cultural, humanities-based, perspective, but relatively little with scientific data, and rarely in combination. We have addressed the unusual location of twenty inhumations found in the sacred heart of the Etruscan city of Tarquinia, focusing on six of these as illustrative, contrasting with the typical contemporary cremations found in cemeteries on the edge of the city. The cultural evidence suggests that the six skeletons were also distinctive in their ritualization and memorialisation. Focusing on the six, as a representative sample, the scientific evidence of osteoarchaeology, isotopic compositions, and ancient DNA has established that these appear to show mobility, diversity and violence through an integrated bioarchaeological approach. The combination of multiple lines of evidence makes major strides towards a deeper understanding of the role of these extraordinary individuals in the life of the early city of Etruria.

Persistence of spermatozoa on decomposing human skin: a scanning electron microscopy study.

Finding spermatozoa is of the utmost importance in judicial cases involving both the living and the dead; however, most of literature actually deals with inner genitalia and does not take into consideration the chance of external deposition of semen on skin, which is not rare. In addition, the most advanced microscopic technologies such as scanning electron microscopy (SEM) have not been thoroughly investigated within this specific field of research. This study aims at applying SEM analysis to samples of decomposed skin in order to test its potential in detecting spermatozoa particularly in decomposed cadavers. A sample of skin was obtained at autopsy and divided into two thin strips; one of the samples was used as a negative control. Semen was then taken from a "donor" (with a normal spermiogram) and was spread onto the other skin sample. Every 3 days for the first 15 days (for a total of six samples), a standard slide was prepared from swabs on the treated and control skin and analyzed by standard light microscopy. In addition, every 7 days up to 91 days (3 months circa), a skin sample was taken from the positive and negative control and examined by SEM for a total of 14 samples. Results show that after 12 days, light microscopy failed in detecting spermatozoa, whereas they were still visible up to 84 days by SEM analysis. This study therefore suggests the persistence of sperm structures in time and in decomposing material as well as the possible application of SEM technology to decomposed skin in order to detect semen.

The application of cone-beam CT in the aging of bone calluses: a new perspective?

In the forensic and anthropological fields, the assessment of the age of a bone callus can be crucial for a correct analysis of injuries in the skeleton. To our knowledge, the studies which have focused on this topic are mainly clinical and still leave much to be desired for forensic purposes, particularly in looking for better methods for aging calluses in view of criminalistic applications. This study aims at evaluating the aid cone-beam CT can give in the investigation of the inner structure of fractures and calluses, thus acquiring a better knowledge of the process of bone remodeling. A total of 13 fractures (three without callus formation and ten with visible callus) of known age from cadavers were subjected to radiological investigations with digital radiography (DR) (conventional radiography) and cone-beam CT with the major aim of investigating the differences between DR and tomographic images when studying the inner and outer structures of bone healing. Results showed how with cone-beam CT the structure of the callus is clearly visible with higher specificity and definition and much more information on mineralization in different sections and planes. These results could lay the foundation for new perspectives on bone callus evaluation and aging with cone-beam CT, a user-friendly and skillful technique which in some instances can also be used extensively on the living (e.g., in cases of child abuse) with reduced exposition to radiation.

Pitfalls at the root of facial assessment on photographs: a quantitative study of accuracy in positioning facial landmarks.

In the last years, facial analysis has gained great interest also for forensic anthropology. The application of facial landmarks may bring about relevant advantages for the analysis of 2D images by measuring distances and extracting quantitative indices. However, this is a complex task which depends upon the variability in positioning facial landmarks. In addition, literature provides only general indications concerning the reliability in positioning facial landmarks on photographic material, and no study is available concerning the specific errors which may be encountered in such an operation. The aim of this study is to analyze the inter- and intra-observer error in defining facial landmarks on photographs by using a software specifically developed for this purpose. Twenty-four operators were requested to define 22 facial landmarks on frontal view photographs and 11 on lateral view images; in addition, three operators repeated the procedure on the same photographs 20 times (at distance of 24 h). In the frontal view, the landmarks with less dispersion were the pupil, cheilion, endocanthion, and stomion (sto), and the landmarks with the highest dispersion were gonion, zygion, frontotemporale, tragion, and selion (se). In the lateral view, the landmarks with the least dispersion were se, pronasale, subnasale, and sto, whereas landmarks with the highest dispersion were gnathion, pogonion, and tragion. Results confirm that few anatomical points can be defined with the highest accuracy and show the importance of the preliminary investigation of reliability in positioning facial landmarks.

The risk of misinterpreting genital signs of sexual abuse in cadavers: a case report.

The significance of genital findings in a case of suspected child sexual abuse has been widely debated in the past decades, as shown by the different classifications available in literature. In the case of postmortem examination, the search for signs of sexual abuse is considerably more difficult because of the superimposition of postmortem modifications, which may determine tissue modifications that can be mistaken for traumatic lesions. This study aims at reporting a case where presumed findings of the first autopsy were denied by histological analysis; in detail, what looked like a possible bruise of the hymen was correctly recognized as hypostasis (livor) of the hymenal tissue by histological analysis. This case report suggests caution in the analysis and discussion of genital lesions found during postmortem examination since the superimposition of cadaveric modifications may radically modify the morphology of soft tissues.

Antidepressant discontinuation syndrome: A state-of-the-art clinical review.

Antidepressant drugs are prescribed to patients with depressive, anxiety disorders, and other conditions. Evidence about antidepressant discontinuation syndrome (ADS) and related outcomes is sparse, although potentially burdensome in some patients. The present state-of-the-art review aims to appraise the most current evidence about ADS critically. ADS has been documented for most antidepressant drugs, although most literature focuses on selective serotonin reuptake inhibitors prescribed for depression. While down-titration cannot exclude the chance of ADS, it is nonetheless warranted in the clinical setting, especially for short half-life and sedative compounds such as paroxetine. Integrative management with concurrent pharmacotherapy and psychotherapy may minimize the eventual unpleasant effects arising within the discontinuation process. In addition, patient-tailored interventions and education should be part of the discontinuation strategy. Future research must rely on broadly accepted definitions for ADS and related phenomena such as antidepressant withdrawal and shed further light on the underpinning neurobiology. Discriminating between ADS-related phenomena and relapse of depression is likewise warranted, along with a neuroscience-based nomenclature instead of a class one.