Exploring adolescent mental health during the COVID-19 crisis - strengths and difficulties.

Introduction: The SARS-CoV-2 pandemic has significantly impacted children and adolescents, leading to mental health challenges. Knowledge on their resources and difficulties is crucial and there is a need for valid instruments to assess their psychosocial condition especially in this exceptional situation. We assessed psychopathological symptoms using the SDQ during the pandemic, comparing to pre-pandemic data. Our study aims to understand adolescents' strengths and difficulties amidst COVID-19, evaluating the SDQ's utility in crisis settings. Methods: Within the German school-based surveillance study ("B-Fast"), we assessed behavioral strengths and difficulties in 664 adolescents aged 11-17 years during the peak of the German COVID-19 pandemic using the validated Strengths and Difficulties Questionnaire (SDQ) for both external and self-assessed data collection. Data were collected between November 2020 and April 2021. We compared self-assessed SDQ-scores to pre-pandemic data from a comparable sample and examined adolescent classification as "normal" or "borderline/abnormal" based on both external and self-assessed SDQ subscale scores using established cut-off values. Additionally, we conducted sex and rater-based score comparisons. Results: In our study, we observed a significant worsening of "Emotional Symptoms" compared to pre-pandemic levels, while "Conduct Problems" and "Prosocial Behavior" showed improvement. Variations in classification to "normal" and "abnormal" emerged when applying German versus British cut-off values. Females scored higher on "Emotional Symptoms" while males scored higher on "Hyperactivity Symptoms." Correlations between external and self-assessed SDQ ratings ranged from 0.43 (p < 0.001) for "Prosocial Behavior" among girls to 0.62 (p < 0.001) for "Peer Problems" among boys, indicating moderate to high consistency. Discussion/conclusion: Our study contributes to understanding the psychosocial impact of the COVID-19 pandemic on German adolescents. Compared to other symptoms, we observed a particular worsening in "Emotional Symptoms" based on our data. Despite the moderate correlation between parental and self-reported evaluations, there appears to be a certain discrepancy in the perception of adolescent quality of life. Therefore, it seems prudent to assess both the external and self-reported evaluations and amalgamate the results from both parties to obtain a comprehensive problem profile of the individual. These findings underscore the importance of using country-specific cutoff values and reaffirm the utility of the SDQ as a valuable assessment tool, even within the unique circumstances posed by a pandemic.

Understanding recessive disease risk in multi-ethnic populations with different degrees of consanguinity.

Population medical genetics aims at translating clinically relevant findings from recent studies of large cohorts into healthcare for individuals. Genetic counseling concerning reproductive risks and options is still mainly based on family history, and consanguinity is viewed to increase the risk for recessive diseases regardless of the demographics. However, in an increasingly multi-ethnic society with diverse approaches to partner selection, healthcare professionals should also sharpen their intuition for the influence of different mating schemes in non-equilibrium dynamics. We, therefore, revisited the so-called out-of-Africa model and studied in forward simulations with discrete and not overlapping generations the effect of inbreeding on the average number of recessive lethals in the genome. We were able to reproduce in both frameworks the drop in the incidence of recessive disorders, which is a transient phenomenon during and after the growth phase of a population, and therefore showed their equivalence. With the simulation frameworks, we also provide the means to study and visualize the effect of different kin sizes and mating schemes on these parameters for educational purposes.

Comparison of Outcomes After Arthroscopic Superior Capsule Reconstruction Versus Arthroscopic Partial Repair or Arthroscopic Debridement for Irreparable Rotator Cuff Tears.

Background: Arthroscopic superior capsule reconstruction (SCR), arthroscopic partial repair (PR), and arthroscopic debridement (DB) are valid treatment options for irreparable rotator cuff (RC) tears. Purpose/Hypothesis: The purpose of this study was to compare clinical, functional, and radiological outcomes of arthroscopic SCR with arthroscopic PR and arthroscopic DB in patients with irreparable posterosuperior RC tears. It was hypothesized that SCR would lead to superior clinical and functional outcomes compared with PR or DB. Study Design: Cohort study; Level of evidence, 3. Methods: Clinical and functional outcomes of this single-center retrospective study included range of motion, strength, and the age- and sex-adjusted Constant-Murley score. Patient-reported outcome measures (PROMs) involved the quick Disabilities of the Arm, Shoulder and Hand score, the Subjective Shoulder Value, and the visual analog scale for pain. Graft and repaired tendon integrity was evaluated by magnetic resonance imaging (MRI) at 12 months of follow-up. Results: In total, 57 patients treated with SCR (n = 20), PR (n = 17), and DB (n = 20) were included. The mean clinical follow-up was 33.8 ± 17.9 months. Preoperative clinical and functional characteristics were comparable among the 3 groups. The range of motion and clinical and functional scores of all 3 groups significantly improved from pre- to postoperatively. Postoperative PROMs showed no differences among all 3 study groups. SCR revealed significantly higher postoperative strength compared with PR (P = .001) and DB (P = .004). Postoperative MRI revealed a rerupture in 4 patients with SCR (20%). Postoperative MRI showed a rerupture in 9 patients with PR ( 53%). Fatty muscle infiltration of the supraspinatus and infraspinatus significantly progressed within all 3 study groups in postoperative MRI scans. No clinical and functional differences were observed between intact and reruptured PR. Conclusion: Patients who underwent SCR had better postoperative strength recovery than patients who underwent PR or DB.

Engineering of extracellular matrix from human iPSC-mesenchymal progenitors to enhance osteogenic capacity of human bone marrow stromal cells independent of their age.

Regeneration of bone defects is often limited due to compromised bone tissue physiology. Previous studies suggest that engineered extracellular matrices enhance the regenerative capacity of mesenchymal stromal cells. In this study, we used human-induced pluripotent stem cells, a scalable source of young mesenchymal progenitors (hiPSC-MPs), to generate extracellular matrix (iECM) and test its effects on the osteogenic capacity of human bone-marrow mesenchymal stromal cells (BMSCs). iECM was deposited as a layer on cell culture dishes and into three-dimensional (3D) silk-based spongy scaffolds. After decellularization, iECM maintained inherent structural proteins including collagens, fibronectin and laminin, and contained minimal residual DNA. Young adult and aged BMSCs cultured on the iECM layer in osteogenic medium exhibited a significant increase in proliferation, osteogenic marker expression, and mineralization as compared to tissue culture plastic. With BMSCs from aged donors, matrix mineralization was only detected when cultured on iECM, but not on tissue culture plastic. When cultured in 3D iECM/silk scaffolds, BMSCs exhibited significantly increased osteogenic gene expression levels and bone matrix deposition. iECM layer showed a similar enhancement of aged BMSC proliferation, osteogenic gene expression, and mineralization compared with extracellular matrix layers derived from young adult or aged BMSCs. However, iECM increased osteogenic differentiation and decreased adipocyte formation compared with single protein substrates including collagen and fibronectin. Together, our data suggest that the microenvironment comprised of iECM can enhance the osteogenic activity of BMSCs, providing a bioactive and scalable biomaterial strategy for enhancing bone regeneration in patients with delayed or failed bone healing.

Efficacy and safety of ruxolitinib in patients with newly-diagnosed polycythemia vera: futility analysis of the RuxoBEAT clinical trial of the GSG-MPN study group.

Patients (pts) with polycythemia vera (PV) suffer from pruritus, night sweats, and other symptoms, as well as from thromboembolic complications and progression to post-PV myelofibrosis. Ruxolitinib (RUX) is approved for second-line therapy in high-risk PV pts with hydroxyurea intolerance or resistance. The RuxoBEAT trial (NCT02577926, registered on October 1, 2015, at clinicaltrials.gov) is a multicenter, open-label, two-arm phase-IIb trial with a target population of 380 pts with PV or ET, randomized to receive RUX or best available therapy. This pre-specified futility analysis assesses the early clinical benefit and tolerability of RUX in previously untreated PV pts (6-week cytoreduction was allowed). Twenty-eight patients were randomly assigned to receive RUX. Compared to baseline, after 6 months of treatment, there was a significant reduction of median hematocrit (46 to 41%), the median number of phlebotomies per year (4.0 to 0), and median patient-reported pruritus scores (2 to 1), and a trend for reduced night sweat scores (1.5 to 0). JAK2V617F allele burden, as part of the scientific research program, also significantly decreased. One hundred nine adverse events (AEs) occurred in 24/28 patients (all grade 1 to 3), and no pt permanently discontinued treatment because of AEs. Thus, treatment with ruxolitinib in untreated PV pts is feasible, well-tolerated, and efficient regarding the above-mentioned endpoints.

The Evolution of Reverse Total Shoulder Arthroplasty-From the First Steps to Novel Implant Designs and Surgical Techniques.

PURPOSE OF REVIEW: The purpose of this review is to summarize recent literature regarding the latest design modifications and biomechanical evolutions of reverse total shoulder arthroplasty and their impact on postoperative outcomes. RECENT FINDINGS: Over the past decade, worldwide implantation rates of reverse total shoulder arthroplasty have drastically increased for various shoulder pathologies. While Paul Grammont's design principles first published in 1985 for reverse total shoulder arthroplasty remained unchanged, several adjustments were made to address postoperative clinical and biomechanical challenges such as implant glenoid loosening, scapular notching, or limited range of motion in order to maximize functional outcomes and increase the longevity of reverse total shoulder arthroplasty. However, the adequate and stable fixation of prosthetic components can be challenging, especially in massive osteoarthritis with concomitant bone loss. To overcome such issues, surgical navigation and patient-specific instruments may be a viable tool to improve accurate prosthetic component positioning. Nevertheless, larger clinical series on the accuracy and possible complications of this novel technique are still missing.

Treatment resistance analysis reveals GLUT-1-mediated glucose uptake as a major target of synthetic rocaglates in cancer cells.

Rocaglates are natural compounds that have been extensively studied for their ability to inhibit translation initiation. Rocaglates represent promising drug candidates for tumor treatment due to their growth-inhibitory effects on neoplastic cells. In contrast to natural rocaglates, synthetic analogues of rocaglates have been less comprehensively characterized, but were also shown to have similar effects on the process of protein translation. Here, we demonstrate an enhanced growth-inhibitory effect of synthetic rocaglates when combined with glucose anti-metabolite 2-deoxy-D-glucose (2DG) in different cancer cell lines. Moreover, we unravel a new aspect in the mechanism of action of synthetic rocaglates involving reduction of glucose uptake mediated by downregulation or abrogation of glucose transporter GLUT-1 expression. Importantly, cells with genetically induced resistance to synthetic rocaglates showed substantially less pronounced treatment effect on glucose metabolism and did not demonstrate GLUT-1 downregulation, pointing at the crucial role of this mechanism for the anti-tumor activity of the synthetic rocaglates. Transcriptome profiling revealed glycolysis as one of the major pathways differentially regulated in sensitive and resistant cells. Analysis of synthetic rocaglate efficacy in a 3D tissue context with a co-culture of tumor and normal cells demonstrated a selective effect on tumor cells and substantiated the mechanistic observations obtained in cancer cell lines. Increased glucose uptake and metabolism is a universal feature across different tumor types. Therefore, targeting this feature by synthetic rocaglates could represent a promising direction for exploitation of rocaglates in novel anti-tumor therapies.

Age-dependent performance of BRAF mutation testing in Lynch syndrome diagnostics.

BRAF V600E mutations have been reported as a marker of sporadic microsatellite instability (MSI) colorectal cancer (CRC). Current international diagnostic guidelines recommend BRAF mutation testing in MSI CRC patients to predict low risk of Lynch syndrome (LS). We evaluated the age-specific performance of BRAF testing in LS diagnostics. We systematically compared the prevalence of BRAF mutations in LS-associated CRCs and unselected MSI CRCs in different age groups as available from published studies, databases and population-based patient cohorts. Sensitivity/specificity analysis of BRAF testing for exclusion of LS and cost calculations were performed. Among 969 MSI CRCs from LS carriers in the literature and German HNPCC Consortium, 15 (1.6%) harbored BRAF mutations. Six of seven LS patients with BRAF-mutant CRC and reported age were <50 years. Among 339 of 756 (44.8%) of BRAF mutations detected in unselected MSI CRC, only 2 of 339 (0.6%) BRAF mutations were detected in patients <50 years. The inclusion of BRAF testing led to high risk of missing LS patients and increased costs at age <50 years. BRAF testing in patients <50 years carries a high risk of missing a hereditary cancer predisposition and is cost-inefficient. We suggest direct referral of MSI CRC patients <50 years to genetic counseling without BRAF testing.

Mid- to Long-Term Outcomes After Deep Infections After Arthroscopic Rotator Cuff Repair.

PURPOSE: To review clinical and subjective outcomes in patients with deep infections after arthroscopic rotator cuff repair. METHODS: All patients in whom deep infections developed after arthroscopic rotator cuff repair at a single center between 2002 and 2016 were retrospectively reviewed. Demographic data, clinical and microbiological findings, and treatment were analyzed. Clinical and subjective outcomes included the Constant score, visual analog scale score for pain, American Shoulder and Elbow Surgeons score, and Simple Shoulder Test score. RESULTS: Thirty patients could be identified and included in the study. The most commonly isolated pathogens were Staphylococcus epidermidis (36.7%) and Cutibacterium acnes (30.0%). In 26 of 30 patients (86.7%), the infection was treated initially with an open surgical approach, whereas 4 patients (13.3%) underwent arthroscopic revision. A transosseous rotator cuff repair could be performed in 20.0% of patients. A single reoperation was sufficient in 80% of patients, whereas 13.3% required 2 revisions and 6.7% required 3. At the final follow-up of 8.3 years (range, 4-14 years), 26 patients (1 woman and 25 men) were available for outcome evaluation. Significant improvement from the initial surgical procedure to final follow-up was detected in the Constant score (25.7 vs 65.7, P < .001), visual analog scale score for pain (7.0 vs 1.7, P < .001), American Shoulder and Elbow Surgeons score (38.0 vs 76.7, P < .001), and Simple Shoulder Test score (4.0 vs 8.3, P < .001). CONCLUSIONS: Patients with deep infections after arthroscopic rotator cuff repair showed moderate mid- to long-term outcomes. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Glenohumeral Microfracturing of Contained Glenohumeral Defects: Mid- to Long-term Outcome.

PURPOSE: To report mid- to long-term clinical and radiological outcomes after microfracturing for symptomatic chondral defects of the glenohumeral joint. METHODS: All patients who underwent glenohumeral arthroscopic microfracturing between 2002 and 2012 at a single center were considered for inclusion in this retrospective study. Clinical outcome was evaluated using the Constant Score, Oxford Shoulder Score, and Subjective Shoulder Value. Progression of joint space narrowing, sclerosis, marginal osteophytes, and presence of cysts over time were assessed using 4 different radiological grading systems. RESULTS: A total of 16 patients (n = 9 female, n = 7 male) with a mean age of 51.8 ± 12.6 years at the time of surgery and a mean follow-up of 122 ± 51.2 months (range, 61-204 months) were included in this retrospective study. Nine patients (56.3%) showed an isolated chondral defect, while 7 patients (43.8%) had concomitant pathologies. Constant Score (60.3 ± 12.7 vs. 85.9 ± 9.3; P < .001), Oxford Shoulder Score (29.0 ± 5.8 vs. 42.4 ± 4.5; P < .001), and Subjective Shoulder Value (23.9 ± 7.4 vs. 84.3 ± 10.9; P < .001) changed significantly from pre- to postoperative. The majority of patients (88%) were able to return to their preoperative level of activity. Three patients (19.8%) developed radiological signs of progressive glenohumeral degeneration during the study period. However, only 1 patient (6.25%) showed a progression of arthritic changes of more than 1 grade according to radiographic classifications. Two patients (12.5%) underwent revision surgery to a hemi shoulder arthroplasty during the study period at 12 and 36 months after the initial procedure. CONCLUSIONS: Glenohumeral microfracturing is commonly performed together with other procedures, but seems to be a feasible treatment option for contained cartilage lesions in active patients reproducibly yielding good mid- to long-term outcome. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Yoga in school sports improves functioning of autonomic nervous system in young adults: A non-randomized controlled pilot study.

BACKGROUND: Yoga in school is a beneficial tool to promote the good health and well-being of students by changing the way they react to stress. The positive effects of yoga-taught in schools-on children, youth and young adults have been demonstrated in former studies using mostly subjective psychometric data. AIM: The present trial aims to evaluate the potential effects of yoga on autonomic regulation in young adults by analyzing heart rate variability (HRV). METHODS: This study is a non-randomized, explorative, two-arm-pilot study with an active control group. Fourteen healthy young adults took part in a 10-week yoga program (90 min once a week) in school and were compared to a control group of 11 students who participated in conventional school sports (90 min once a week over 10 weeks). 24-hour electrocardiograms (ECGs) were recorded at baseline and following the 10-week intervention. From 20-minute of nocturnal sleep phases, HRV parameters were calculated from linear (time and frequency domain) and nonlinear dynamics (such as symbolic dynamics and Poincaré plot analysis). Analyses of variance (ANOVA) followed by t-tests as post-hoc tests estimating both statistical significance and effect size were used to compare pre-post-intervention for the two groups. RESULTS: The statistical analysis of the interaction effects did not reveal a significant group and time interaction for the individual nocturnal HRV indices. Almost all indices revealed medium and large effects regarding the time main effects. The changes in the HRV indices following the intervention were more dramatic for the yoga group than for the control group which is reflected in predominantly higher significances and stronger effect sizes in the yoga group. CONCLUSION: In this explorative pilot trial, an increase of HRV (more parasympathetic dominance and overall higher HRV) after ten weeks of yoga in school in comparison to regular school sports was demonstrated, showing an improved self-regulation of the autonomic nervous system.

Mildly decreased preoperative bilirubin levels are associated with infections after shoulder and knee arthroplasty.

Increasing numbers of arthroplasties are also accompanied by postoperative infections. The main purpose was to evaluate preoperative serum bilirubin levels between patients with and without infections after shoulder and knee arthroplasties. For this retrospective case-control single-center study, a total of 108 patients were extracted from a prospectively collected database. Eighteen patients with infections after shoulder (n = 8) and knee (n = 10) arthroplasty were matched by age, gender, and implant type in a 1:5-scenario to 90 patients (40 shoulders and 50 knees) without postoperative infection. Demographic data, preoperative blood parameters, and postoperative infection-related outcomes were evaluated. Total bilirubin was the only preoperative parameter significantly different between the infection (8.21 ± 3.25 µmol/L or 0.48 ± 0.19 mg/dL) and noninfection (10.78 ± 4.62 µmol/L or 0.63 ± 0.27 mg/dL; P = .014) group, while C-reactive protein and other liver parameters were similar between the groups. Significantly more controls (92.1%) had preoperative bilirubin levels above 8.72 µmol/L or 0.51 mg/dL than cases (7.9%; P = .007). The 5-year infection survival-rate was 65.6% for patients with preoperative bilirubin levels < 8.72 µmol/L or < 0.51 mg/dL and 91.2% with ≥ 8.72 µmol/L or ≥ 0.51 mg/dL. Mildly decreased preoperative bilirubin levels with a cutoff at 8.72 µmol/L or 0.51 mg/dL were significantly associated to patients with infections after shoulder and knee arthroplasty. There were no differences in other blood parameters or comorbidities between patients with infections and their matched-controls.

MicroRNA Profiling Reveals Distinct Signatures in Degenerative Rotator Cuff Pathologies.

MicroRNAs (miRNAs) have emerged as key regulators orchestrating a wide range of inflammatory and fibrotic diseases. However, the role of miRNAs in degenerative shoulder joint disorders is poorly understood. The aim of this explorative case-control study was to identify pathology-related, circulating miRNAs in patients with chronic rotator cuff tendinopathy and degenerative rotator cuff tears (RCT). In 2017, 15 patients were prospectively enrolled and assigned to three groups based on the diagnosed pathology: (i) no shoulder pathology, (ii) chronic rotator cuff tendinopathy, and (iii) degenerative RCTs. In total, 14 patients were included. Venous blood samples ("liquid biopsies") were collected from each patient and serum levels of 187 miRNAs were determined. Subsequently, the change in expression of nine candidate miRNAs was verified in tendon biopsy samples, collected from patients who underwent arthroscopic shoulder surgery between 2015 and 2018. Overall, we identified several miRNAs to be progressively deregulated in sera from patients with either chronic rotator cuff tendinopathy or degenerative RCTs. Importantly, for the several of these miRNAs candidates repression was also evident in tendon biopsies harvested from patients who were treated for a supraspinatus tendon tear. As similar expression profiles were determined for tendon samples, the newly identified systemic miRNA signature has potential as novel diagnostic or prognostic biomarkers for degenerative rotator cuff pathologies. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. Inc. J Orthop Res 38:202-211, 2020.

Patient-specific instrumentation improved three-dimensional accuracy in total knee arthroplasty: a comparative radiographic analysis of 1257 total knee arthroplasties.

BACKGROUND: The purpose of this study was to compare restoration of mechanical limb alignment and three-dimensional component-positioning between conventional and patient-specific instrumentation in total knee arthroplasty. METHODS: Radiographic data of patients undergoing mobile-bearing total knee arthroplasty (n = 1257), using either conventional (n = 442) or patient-specific instrumentation (n = 812), were analyzed. To evaluate accuracy of axis restoration and 3D-component-positioning between conventional and patient-specific instrumentation, absolute deviations from the targeted neutral mechanical limb alignment and planned implant positions were determined. Measurements were performed on standardized coronal long-leg and sagittal knee radiographs. CT-scans were evaluated for accuracy of axial femoral implant rotation. Outliers were defined as deviations from the targeted neutral mechanical axis of > ± 3° or from the intraoperative component-positioning goals of > ± 2°. Deviations greater than ± 5° from set targets were considered to be severe outliers. RESULTS: Deviations from a neutral mechanical axis (conventional instrumentation: 2.3°± 1.7° vs. patient-specific instrumentation: 1.7°± 1.2°; p < 0.001) and numbers of outliers (conventional instrumentation: 25.8% vs. patient-specific instrumentation: 10.1%; p < 0.001) were significantly lower in the patient-specific instrumentation group. Significantly lower mean deviations and less outliers were detected regarding 3D-component-positioning in the patient-specific instrumentation compared to the conventional instrumentation group (all p < 0.05). CONCLUSIONS: Patient-specific instrumentation prevented from severe limb malalignment and component-positioning outliers (> ± 5° deviation). Use of patient-specific instrumentation proved to be superior to conventional instrumentation in achieving more accurate limb alignment and 3D-component positioning, particularly regarding femoral component rotation. Furthermore, the use of patient-specific instrumentation successfully prevented severe (> 5° deviation) outliers.

Author Correction: Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

External validation of molecular subtype classifications of colorectal cancer based on microsatellite instability, CIMP, BRAF and KRAS.

BACKGROUND: Competing molecular classification systems have been proposed to complement the TNM staging system for a better prediction of survival in colorectal cancer (CRC). However, validation studies are so far lacking. The aim of this study was to validate and extend previously published molecular classifications of CRC in a large independent cohort of CRC patients. METHODS: CRC patients were recruited into a population-based cohort study (DACHS). Molecular subtypes were categorized based on three previously published classifications. Cox-proportional hazard models, based on the same set of patients and using the same confounders as reported by the original studies, were used to determine overall, cancer-specific, or relapse-free survival for each subtype. Hazard ratios and confidence intervals, as well as Kaplan-Meier plots were compared to those reported by the original studies. RESULTS: We observed similar patterns of worse survival for the microsatellite stable (MSS)/BRAF-mutated and MSS/KRAS-mutated subtypes in our validation analyses, which were included in two of the validated classifications. Of the two MSI subtypes, one defined by additional presence of CIMP-high and BRAF-mutation and the other by tumors negative for CIMP, BRAF and KRAS-mutations, we could not confirm associations with better prognosis as suggested by one of the classifications. For two of the published classifications, we were able to provide results for additional subgroups not included in the original studies (men, other disease stages, other locations). CONCLUSIONS: External validation of three previously proposed classifications confirmed findings of worse survival for CRC patients with MSS subtypes and BRAF or KRAS mutations. Regarding MSI subtypes, other patient characteristics such as stage of the tumor, may influence the potential survival benefit. Further integration of methylation, genetic, and immunological information is needed to develop and validate a comprehensive classification that will have relevance for use in clinical practice.

Chloroplast-localized BICAT proteins shape stromal calcium signals and are required for efficient photosynthesis.

The photosynthetic machinery of plants must be regulated to maximize the efficiency of light reactions and CO2 fixation. Changes in free Ca2+ in the stroma of chloroplasts have been observed at the transition between light and darkness, and also in response to stress stimuli. Such Ca2+ dynamics have been proposed to regulate photosynthetic capacity. However, the molecular mechanisms of Ca2+ fluxes in the chloroplasts have been unknown. By employing a Ca2+ reporter-based approach, we identified two chloroplast-localized Ca2+ transporters in Arabidopsis thaliana, BICAT1 and BICAT2, that determine the amplitude of the darkness-induced Ca2+ signal in the chloroplast stroma. BICAT2 mediated Ca2+ uptake across the chloroplast envelope, and its knockout mutation strongly dampened the dark-induced [Ca2+ ]stroma signal. Conversely, this Ca2+ transient was increased in knockout mutants of BICAT1, which transports Ca2+ into the thylakoid lumen. Knockout mutation of BICAT2 caused severe defects in chloroplast morphology, pigmentation and photosynthetic light reactions, rendering bicat2 mutants barely viable under autotrophic growth conditions, while bicat1 mutants were less affected. These results show that BICAT transporters play a role in chloroplast Ca2+ homeostasis. They are also involved in the regulation of photosynthesis and plant productivity. Further work will be required to reveal whether the effect on photosynthesis is a direct result of their role as Ca2+ transporters.

Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.

Extracellular vesicles (EVs) are natural nanoparticles that play important roles in intercellular communication and are increasingly studied for biosignalling, pathogenesis and therapy. Nevertheless, little is known about optimal conditions for their transfer and storage, and the potential impact on preserving EV-loaded cargoes. We present the first comprehensive stability assessment of different widely available types of EVs during various storage conditions including -80 °C, 4 °C, room temperature, and freeze-drying (lyophilisation). Lyophilisation of EVs would allow easy handling at room temperature and thus significantly enhance their expanded investigation. A model enzyme, ß-glucuronidase, was loaded into different types of EVs derived from mesenchymal stem cells, endothelial cells and cancer cells. Using asymmetric flow field-flow fractionation we proved that the model enzyme is indeed stably encapsulated into EVs. When assessing enzyme activity as indicator for EV stability, and in comparison to liposomes, we show that EVs are intrinsically stable during lyophilisation, an effect further enhanced by cryoprotectants. Our findings provide new insight for exploring lyophilisation as a novel storage modality and we create an important basis for standardised and advanced EV applications in biomedical research.