Structure of the connexin-43 gap junction channel in a putative closed state

Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state

Comprehensive Overview of Bottom-up Proteomics using Mass Spectrometry

Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods to aid the novice and experienced researcher. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this work to serve as a basic resource for new practitioners in the field of shotgun or bottom-up proteomics

Contrast-Enhanced Ultrasound with VEGFR2-Targeted Microbubbles for Monitoring Regorafenib Therapy Effects in Experimental Colorectal Adenocarcinomas in Rats with DCE-MRI and Immunohistochemical Validation

Objectives To investigate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring therapy effects of regorafenib on experimental colon carcinomas in rats with correlation to dynamic contrast-enhanced MRI (DCE-MRI) and immunohistochemistry. Materials and Methods: Human colorectal adenocarcinoma xenografts (HT-29) were implanted subcutaneously in n =21 (n = 11 therapy group;n = 10 control group) female athymic nude rats (Hsd: RH-Foxn1 (mu)). Animals were imaged at baseline and after a one-week daily treatment with regorafenib or a placebo (10 mg/kg bodyweight), using CEUS with VEGFR2-targeted microbubbles and DCE-MRI. In CEUS tumor perfusion was assessed during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity after 8 (SI8min) and 10 minutes (SI10min)), using a conventional 15L8 linear transducer (transmit frequency 7 MHz, dynamic range 80 dB, depth 25 mm). In DCE-MRI functional parameters plasma flow (PF) and plasma volume (PV) were quantified. For validation purposes, CEUS parameters were correlated with DCE-MRI parameters and immunohistochemical VEGFR2, CD31, Ki-67 and TUNEL stainings. Results: CEUS perfusion parameter WiAUC decreased significantly (116,989 +/- 77,048 a.u. to 30,076 +/- 27,095a.u.;p = 0.005) under therapy with no significant changes (133,932 +/- 65,960 a.u. to 84,316 +/- 74,144 a.u.;p = 0.093) in the control group. In the therapy group, the amount of bound microbubbles in the late phase was significantly lower in the therapy than in the control group on day 7 (SI8min: 283 +/- 191 vs. 802 +/- 460 a.u.;p = 0.006);SI10min: 226 +/- 149 vs. 645 +/- 461 a.u.;p = 0.009). PF and PV decreased significantly (PF: 147 +/- 58 mL/100 mL/min to 71 +/- 15 mL/100 mL/min;p = 0.003;PV: 13 +/- 3% to 9 +/- 4%;p = 0.040) in the therapy group. Immunohistochemistry revealed significantly fewer VEGFR2 (7.2 +/- 1.8 vs. 17.8 +/- 4.6;p < 0.001), CD31 (8.1 +/- 3.0 vs. 20.8 +/- 5.7;p < 0.001) and Ki-67 (318.7 +/- 94.0 vs. 468.0 +/- 133.8;p = 0.004) and significantly more TUNEL (672.7 +/- 194.0 vs. 357.6 +/- 192.0;p = 0.003) positive cells in the therapy group. CEUS parameters showed significant (p < 0.05) correlations to DCE-MRI parameters and immunohistochemistry. Conclusions CEUS with VEGFR2-targeted microbubbles allowed for monitoring regorafenib functional and molecular therapy effects on experimental colorectal adenocarcinomas with a significant decline of CEUS and DCE-MRI perfusion parameters as well as a significant reduction of specifically bound microbubbles under therapy, consistent with a reduced expression of VEGFR2

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Coming to terms with oneself: a mixed methods approach to perceived self-esteem of adult survivors of childhood maltreatment in foster care settings

Abstract Background A broad range of psychopathological sequelae was found in adult survivors of institutional childhood maltreatment (IM). Childhood maltreatment is also associated with lower self–esteem (SE). In previous qualitative research, adult survivors of IM reported feelings of worthlessness and self-doubts, but research on IM and its associations with SE is still scarce. Method To investigate the emotional facet of SE in 46 adult survivors of IM in foster care settings provided by the City of Vienna we used the Emotional SE subscale of the Multidimensional Self-Esteem Scale (‘Multidimensionale Selbstwertskala’, MSWS) and applied a semi-structured interview with open-ended questions. Qualitative data were analyzed with thematic analysis. Finally, qualitative and quantitative data were merged in a mixed method approach to detect similarities and differences between both assessment modalities. Results Findings showed a significantly lower emotional SE level (MSWS) in adult survivors compared to a norm sample. Qualitative findings revealed five main themes reporting positive and negative emotions and attitudes towards oneself. Merged data showed a tendency of more positive attitudes and emotions within participants with higher emotional SE levels and more negative attitudes within participants with lower levels. No gender differences were found in both data sets. Conclusions IM seems to predict lower emotional SE. Observed qualitative aspects of emotional SE seem to concur with symptoms of disturbances in self-organization (DSO) that are typically present in persons suffering from Complex PTSD. Considering emotional SE in future research could facilitate the understanding of the sequelae of complex trauma

Self-perceived problems of Afghan asylum seekers and refugees and their experiences with a short psychological intervention

Abstract Background The present study examined Afghan asylum seekers’ and refugees’ self-perceived problems, and their experiences of treatment with an adapted version of the brief transdiagnostic psychological intervention “Problem Management Plus” (aPM+). Specifically, the study explored which problems trouble them most and how these problems influence their daily functioning. Further, it examined how various standardized outcome measures correlate with these subjectively perceived problems. Method This study is part of a larger RCT study (PIAAS study) in which 88 Afghan asylum seekers and refugees were randomly allocated either to aPM + in addition to treatment as usual (aPM+/TAU) or TAU alone. The presented study uses a multi-method approach consisting of two parts: First, we investigated participants’ self-identified problems and subjective functional impairment using quantitative and qualitative assessment in both the aPM+/TAU and TAU group (n = 88). Second, we conducted in-depth qualitative interviews with a subsample of the aPM+/TAU group (n = 24) to gain a deeper understanding of participants’ personal experiences with aPM + and to obtain suggestions for improvement. Spearman correlations were applied for quantitative data, and deductive and inductive approaches of thematic analysis were used for qualitative data. Results We identified six main themes of self-perceived problems (primary post-migration living difficulties, general mental health problems, interpersonal stressors, secondary post-migration living difficulties, mental health problems specifically associated with stress, and somatic problems) and their consequences, as well as subjective functional impairment. Standardized measures of general mental health, posttraumatic psychopathology, and quality of life did not correlate with the intensity of self-perceived problems. aPM + was mostly perceived positively, and few participants had recommendations for its improvement. Conclusion The study aimed at giving a voice to Afghan trauma survivors to inform service providers and policy makers about their needs. Based on their expertise, future interventions can be tailored to their actual needs and optimized in terms of practical use. aPM + proved to be a positively perceived intervention that reduces subjective symptom burden and facilitates daily functioning. Culture-sensitive treatments within (mental) health services should increase service utilization and improve (mental) health in the long term

Complex post-traumatic stress disorder and post-migration living difficulties in traumatised refugees and asylum seekers: the role of language acquisition and barriers

Background: Numerous traumatic experiences and post-migration living difficulties (PMLDs) increase the risk of developing symptoms of complex post-traumatic stress disorder (CPTSD) among Afghan refugees and asylum seekers, living in Austria. Research has repeatedly associated higher levels of CPTSD with higher levels of PMLDs. Summarizing PMLDs into empirically derived factors might facilitate a further understanding of their interaction with symptom presentation within distinct clusters of CPTSD. Objective: The current study aimed to investigate homogeneous subgroups of ICD-11 CPTSD and their association with demographic variables, traumatic experiences, and empirically derived factors of PMLDs. Method: Within a randomized controlled trail (RCT) CPTSD, PMLDs, and traumatic experiences were assessed in a sample of 93 treatment-seeking Afghan refugees and asylum seekers through a fully structured face-to-face and interpreter-assisted interview using the ITQ, the PMLDC, and a trauma checklist. Underlying clusters of CPTSD, superior factors of PMLDs, and their associations were investigated. Results: In total, 19.4% of the sample met the diagnostic criteria for PTSD and 49.5% for CPTSD. We identified a 2-cluster solution consisting of two distinct subgroups as best fitting: (1) a CPTSD cluster and (2) a PTSD cluster. The multitude of PMLDs was summarized into four superior factors. CPTSD cluster membership was associated with childhood potentially traumatic experience types, and one of four PMLD factors, namely ‘language acquisition & barriers’. Conclusions: The results suggest that not PMLDs in general, but rather specific types of PMLDs, are associated with CPTSD. An assumed bidirectional relationship between these PMLD factors and CPTSD symptoms might lead to a downward spiral of increasing distress, and could be considered in treatment strategies

"My brain freezes and I am blocked again": The subjective experience of post-migration living difficulties influenced by complex posttraumatic stress disorder of Afghan asylum seekers and refugees in Austria.

BackgroundPotentially traumatic experiences and post-migration living difficulties (PMLDs) undoubtedly leave marks on mental health and psychosocial functioning. While PMLDs are recognised as a risk factor for posttraumatic stress disorder and complex posttraumatic stress disorder (described together here as C/PTSD), recent investigations have found that C/PTSD symptoms might also influence the experience of PMLDs. The subjective experience of and coping with PMLDs in the context of C/PTSD symptoms has not yet been explored.MethodsSemi-structured, interpreter-assisted interviews exploring the subjective experience of post-migration living difficulties were conducted with treatment-seeking Afghan refugees and asylum seekers (N = 24) and transcribed verbatim. Participants were screened using the International Trauma Questionnaire (ITQ) and allocated to a C/PTSD group or non-C/PTSD group. We analysed the qualitative interviews using content analysis and then compared the results of the two groups.ResultsOver half of the participants (58.3%) met the criteria for C/PTSD. While the two groups addressed numerous similar themes, the C/PTSD group more frequently mentioned themes associated with C/PTSD symptoms (e.g., intrusions, avoidance, sleep disturbances, affective dysregulation) that influenced their responses to PMLDs. The non-C/PTSD group more often experienced positive emotions such as gratitude and optimism, and showed more active, solution-oriented behaviour as well as positive self-verbalisation.ConclusionTo achieve a deeper understanding of PMLDs, post-traumatic psychopathology should be taken into account, as C/PTSD symptoms influence the experience of and coping with PMLDs. The specifics of individual experiences need to be considered in order to promote adaptive coping with PMLDs and to set individual trauma-focused and transdiagnostic treatment targets. In addition, psychological interventions should incorporate psychoeducation to improve the understanding of the impact of C/PTSD on the current experience of PMLDs

Institutional abuse of children in the Austrian Catholic Church: Types of abuse and impact on adult survivors’ current mental health

Celem badania była ocena charakteru i zakresu instytucjonalnego krzywdzenia dzieci przez austriacki Kościół katolicki oraz rozpoznanie obecnego stanu zdrowia psychicznego dorosłych, którzy w dzieciństwie padli ofiarą takiego krzywdzenia. Dane zbierano dwuetapowo. W pierwszej kolejności przeanalizowano dokumentację 448 dorosłych ofiar (M = 55,1 lat, 75,7% mężczyźni), które ujawniły doznane w dzieciństwie krzywdzenie, zeznając przed komisją ds. ochrony ofiar. Uwzględniono różne typy krzywdzenia, różne rodzaje sprawców i rodzinne czynniki ryzyka. W kolejnym etapie grupa 185 dorosłych ofiar została poproszona o wypełnienie kwestionariusza PCL-C (Posttraumatic Stress Disorder Checklist) oraz skróconego inwentarza objawów BSI (Brief Symptom Inventory). Uczestnicy opisywali ogromną różnorodność aktów przemocy fizycznej, seksualnej i emocjonalnej, do których doszło w okresie ich dzieciństwa. Większość ofiar (83,3%) doświadczyła w dzieciństwie krzywdzenia emocjonalnego. Odsetki dotyczące krzywdzenia seksualnego (68,8%) oraz fizycznego (68,3%) były prawie równie wysokie. Częstość występowania zespołu stresu pourazowego (PTSD) wyniosła 48,6%, a u 84,9% ofiar stwierdzono istotne klinicznie objawy w przynajmniej jednej z 10 kategorii objawów (dziewięć podskal BSI oraz PTSD). Nie udało się wyodrębnić konkretnego czynnika obecnego przed wystąpieniem krzywdzenia, który determinowałby rozwój PTSD w późniejszym życiu (np. ubóstwo, przemoc domowa). Zauważono jednak, że ofiary z rozpoznanym PTSD zgłaszały istotnie większą całkowitą liczbę rodzinnych czynników ryzyka (d = 0,33). Autorzy konkludują, że krzywdzenie instytucjonalne w dzieciństwie obejmuje szeroki zakres aktów przemocy, i bardzo silnie ujemnie wpływa na stan zdrowia psychicznego dorosłych, którzy jako dzieci takiego krzywdzenia doświadczyli. Artykuł odnosi się do długoterminowych skutków traumatycznych zdarzeń, nawiązuje też do odnowienia traumy w wieku dorosłym, ponieważ obydwa te zjawiska mogą być trudnym wyzwaniem dla profesjonalistów pracujących z ofiarami.The aim of this study was to explore the nature and dimensions of institutional child abuse (IA) by the Austrian Catholic Church and to investigate the current mental health of adult survivors. Data were collected in two steps. First, documents of 448 adult survivors of IA (M = 55.1 years, 75.7% men) who had disclosed their abuse history to a victim protection commission were collected. Different types of abuse, perpetrator characteristics, and family related risk factors were investigated. Second, a sample of 185 adult survivors completed the Posttraumatic Stress Disorder Checklist (PCL-C) and the Brief Symptom Inventory (BSI).Participants reported an enormous diversity of acts of violent physical, sexual, and emotional abuse that had occurred in their childhood. The majority of adult survivors (83.3%)experienced emotional abuse. Rates of sexual (68.8%) and physical abuse (68.3%) were almost equally high. The prevalence of PTSD was 48.6% and 84.9% showed clinically relevant symptoms in at least one 1 of 10 symptom dimensions (9 BSI subscales and PTSD).No specific pre-IA influence was found to influence the development of PTSD in later life (e.g. poverty, domestic violence). However, survivors with PTSD reported a significantly higher total number of family related risk factors (d = 0.33). We conclude that childhood IA includes a wide spectrum of violent acts, and has a massive negative impact on the current mental health of adult survivors. We address the long-term effects of these traumatic experiences in addition to trauma re-activation in adulthood as both bear great challenges for professionals working with survivors

Institutional abuse of children in the Austrian Catholic Church: Types of abuse and impact on adult survivors’ current mental health

Celem badania była ocena charakteru i zakresu instytucjonalnego krzywdzenia dzieci przez austriacki Kościół katolicki oraz rozpoznanie obecnego stanu zdrowia psychicznego dorosłych, którzy w dzieciństwie padli ofiarą takiego krzywdzenia. Dane zbierano dwuetapowo. W pierwszej kolejności przeanalizowano dokumentację 448 dorosłych ofiar (M = 55,1 lat, 75,7% mężczyźni), które ujawniły doznane w dzieciństwie krzywdzenie, zeznając przed komisją ds. ochrony ofiar. Uwzględniono różne typy krzywdzenia, różne rodzaje sprawców i rodzinne czynniki ryzyka. W kolejnym etapie grupa 185 dorosłych ofiar została poproszona o wypełnienie kwestionariusza PCL-C (Posttraumatic Stress Disorder Checklist) oraz skróconego inwentarza objawów BSI (Brief Symptom Inventory). Uczestnicy opisywali ogromną różnorodność aktów przemocy fizycznej, seksualnej i emocjonalnej, do których doszło w okresie ich dzieciństwa. Większość ofiar (83,3%) doświadczyła w dzieciństwie krzywdzenia emocjonalnego. Odsetki dotyczące krzywdzenia seksualnego (68,8%) oraz fizycznego (68,3%) były prawie równie wysokie. Częstość występowania zespołu stresu pourazowego (PTSD) wyniosła 48,6%, a u 84,9% ofiar stwierdzono istotne klinicznie objawy w przynajmniej jednej z 10 kategorii objawów (dziewięć podskal BSI oraz PTSD). Nie udało się wyodrębnić konkretnego czynnika obecnego przed wystąpieniem krzywdzenia, który determinowałby rozwój PTSD w późniejszym życiu (np. ubóstwo, przemoc domowa). Zauważono jednak, że ofiary z rozpoznanym PTSD zgłaszały istotnie większą całkowitą liczbę rodzinnych czynników ryzyka (d = 0,33). Autorzy konkludują, że krzywdzenie instytucjonalne w dzieciństwie obejmuje szeroki zakres aktów przemocy, i bardzo silnie ujemnie wpływa na stan zdrowia psychicznego dorosłych, którzy jako dzieci takiego krzywdzenia doświadczyli. Artykuł odnosi się do długoterminowych skutków traumatycznych zdarzeń, nawiązuje też do odnowienia traumy w wieku dorosłym, ponieważ obydwa te zjawiska mogą być trudnym wyzwaniem dla profesjonalistów pracujących z ofiarami.The aim of this study was to explore the nature and dimensions of institutional child abuse (IA) by the Austrian Catholic Church and to investigate the current mental health of adult survivors. Data were collected in two steps. First, documents of 448 adult survivors of IA (M = 55.1 years, 75.7% men) who had disclosed their abuse history to a victim protection commission were collected. Different types of abuse, perpetrator characteristics, and family related risk factors were investigated. Second, a sample of 185 adult survivors completed the Posttraumatic Stress Disorder Checklist (PCL-C) and the Brief Symptom Inventory (BSI).Participants reported an enormous diversity of acts of violent physical, sexual, and emotional abuse that had occurred in their childhood. The majority of adult survivors (83.3%)experienced emotional abuse. Rates of sexual (68.8%) and physical abuse (68.3%) were almost equally high. The prevalence of PTSD was 48.6% and 84.9% showed clinically relevant symptoms in at least one 1 of 10 symptom dimensions (9 BSI subscales and PTSD).No specific pre-IA influence was found to influence the development of PTSD in later life (e.g. poverty, domestic violence). However, survivors with PTSD reported a significantly higher total number of family related risk factors (d = 0.33). We conclude that childhood IA includes a wide spectrum of violent acts, and has a massive negative impact on the current mental health of adult survivors. We address the long-term effects of these traumatic experiences in addition to trauma re-activation in adulthood as both bear great challenges for professionals working with survivors