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There are currently no good ways to track human cells in vivo in a clinical setting using magnetic resonance imaging (MRI) based on superparamagnetic iron-oxide nanoparticles. Mya Thu and colleagues have introduced a simple magnetic cell labeling approach that combines three currently US Food and Drug Administration–approved drugs—ferumoxytol, heparin and protamine—to form self-assembling nanocomplexes of about 150 nm in size that effectively label cells for MRI. The approach was shown to effectively label three types of stem cells and two types of immune cells. We report on a new straightforward magnetic cell-labeling approach that combines three US Food and Drug Administration (FDA)-approved drugs—ferumoxytol, heparin and protamine—in serum-free medium to form self-assembling nanocomplexes that effectively label cells for in vivo magnetic resonance imaging (MRI). We observed that the ferumoxytol-heparin-protamine (HPF) nanocomplexes were stable in serum-free cell culture medium. HPF nanocomplexes show a threefold increase in T2 relaxivity compared to ferumoxytol. Electron microscopy showed internalized HPF in endosomes, which we confirmed by Prussian blue staining of labeled cells. There was no long-term effect or toxicity on cellular physiology or function of HPF-labeled hematopoietic stem cells, bone marrow stromal cells, neural stem cells or T cells when compared to controls. In vivo MRI detected 1,000 HPF-labeled cells implanted in rat brains. This HPF labeling method should facilitate the monitoring by MRI of infused or implanted cells in clinical trials.

Pharmacological approaches to treat breast cancer metastases in the brain have been met with limited success. In part, the impermeability of the blood brain barrier (BBB) has hindered delivery of chemotherapeutic agents to metastatic tumors in the brain. BBB-permeable chemotherapeutic drugs are being developed, and noninvasively assessing the efficacy of these agents will be important in both preclinical and clinical settings. In this regard, dynamic contrast enhanced (DCE) and diffusion weighted imaging (DWI) are magnetic resonance imaging (MRI) techniques to monitor tumor vascular permeability and cellularity, respectively. In a rat model of metastatic breast cancer, we demonstrate that brain and bone metastases develop with distinct physiological characteristics as measured with MRI. Specifically, brain metastases have limited permeability of the BBB as assessed with DCE and an increased apparent diffusion coefficient (ADC) measured with DWI compared to the surrounding brain. Microscopically, brain metastases were highly infiltrative, grew through vessel co-option, and caused extensive edema and injury to the surrounding neurons and their dendrites. By comparison, metastases situated in the leptomenengies or in the bone had high vascular permeability and significantly lower ADC values suggestive of hypercellularity. On histological examination, tumors in the bone and leptomenengies were solid masses with distinct tumor margins. The different characteristics of these tissue sites highlight the influence of the microenvironment on metastatic tumor growth. In light of these results, the suitability of DWI and DCE to evaluate the response of chemotherapeutic and anti-angiogenic agents used to treat co-opted brain metastases, respectively, remains a formidable challenge.

This qualitative study examined how emerging adults perceived their childhood experiences related to their sibling with depression or anxiety and how they believed it impacted current functioning. Participants were given the opportunity to share their personal experiences. Multiple sampling methods were used for an expansive recruitment process for this thematic qualitative analysis. Nine participants who ranged from ages 22–28 years and included 4 women, 4 men, and one transgender man with varied racial and sexual identities, all completed semi-structured interviews. Data were analyzed using thematic analysis in NVivo where multiple reviews of theme and the codebook were conducted with an external consultant for a review of credibility and trustworthiness. Nine themes described participants experiences through a developmental and family systems perspective. In childhood, participants described feeling a strong shift of focus on their sibling with depression or anxiety, which led them to suppress their own emotions and be a support system to others. The study developed from past research as many participants were able to reflect on positive changes. In adulthood, many described similar negative patterns, however they experienced improvements in boundary setting, growing in perspective taking, and relationship skills. Meaning making developed for most participants in adulthood which created more empathy towards their sibling and other relations which manifested as motivation to support others. Several participants endorsed pursuing a career in mental health with identifying their experience growing up with a sibling with depression or anxiety as a leading factor to this decision. They also had the opportunity to discuss resources that they believe could have been helpful to the family unit when they were growing up. Strengths and limitations of this study were discussed as well as considerations for future studies and reflections on how mental health professionals can support diverse communities with the findings from this research.

We report on a novel and straightforward magnetic cell labeling approach that combines three FDA-approved drugs, ferumoxytol (F), heparin (H) and protamine (P) in serum free media to form self-assembling nanocomplexes that effectively label cells for in vivo MRI. We observed that the HPF nanocomplexes were stable in serum free cell culture media. HPF nanocomplexes exhibited a three-fold increase in T2 relaxivity compared to F. Electron Microscopy revealed internalized HPF within endosomes, confirmed by Prussian blue staining of labeled cells. There was no long-term effect or toxicity on cellular physiology or function of HPF-labeled hematopoietic stem cells, bone marrow stromal cells, neural stem cells, and T-cells when compared to controls. In vivo MRI detected 1000 HPF-labeled cells implanted in rat brains. HPF labeling method should facilitate the monitoring by MRI of infused or implanted cells in clinical trials.

Magnetic resonance imaging (MRI) has detected changes in pancreas volume and other characteristics in type 1 and type 2 diabetes. However, differences in MRI technology and approaches across locations currently limit the incorporation of pancreas imaging into multisite trials. The purpose of this study was to develop a standardized MRI protocol for pancreas imaging and to define the reproducibility of these measurements. Calibrated phantoms with known MRI properties were imaged at five sites with differing MRI hardware and software to develop a harmonized MRI imaging protocol. Subsequently, five healthy volunteers underwent MRI at four sites using the harmonized protocol to assess pancreas size, shape, apparent diffusion coefficient (ADC), longitudinal relaxation time (T1), magnetization transfer ratio (MTR), and pancreas and hepatic fat fraction. Following harmonization, pancreas size, surface area to volume ratio, diffusion, and longitudinal relaxation time were reproducible, with coefficients of variation less than 10%. In contrast, non-standardized image processing led to greater variation in MRI measurements. By using a standardized MRI image acquisition and processing protocol, quantitative MRI of the pancreas performed at multiple locations can be incorporated into clinical trials comparing pancreas imaging measures and metabolic state in individuals with type 1 or type 2 diabetes.

T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker. The potency of this Prot- FOLR1-TCB is recovered following protease-cleavage of the linker releasing the anti-idiotypic anti-CD3 scFv. In vivo, the Prot-FOLR1-TCB mediates antitumor efficacy comparable to the parental FOLR1-TCB whereas a noncleavable control Prot-FOLR1-TCB is inactive. In contrast, killing of bronchial epithelial and renal cortical cells with low FOLR1 expression is prevented compared to the parental FOLR1-TCB. The findings are confirmed for mesothelin as alternative tumor antigen. Thus, masking the anti-CD3 Fab fragment with an anti-idiotypic mask and cleavage of the mask by tumor-specific proteases can be applied to enhance specificity and safety of TCBs. The clinical application of T cell bispecific antibodies (TCBs) is often limited by the lack of tumour-specific antigens. In this study, the authors present a strategy to increase TCB tumour-selectivity by adding an anti-CD3 moiety that can be specifically activated by tumor specific proteases in the tumor microenvironment.

Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation by proteases. In most prokaryotes and in chloroplasts and mitochondria, protein degradation is performed by the caseinolytic protease ClpP, a tetradecamer barrel-like proteolytic complex. Dysregulating ClpP function has shown promise in fighting antibiotic resistance and as a potential therapy for acute myeloid leukemia. Here we use methyl–transverse relaxation-optimized spectroscopy (TROSY)–based NMR, cryo-EM, biochemical assays, and molecular dynamics simulations to characterize the structural dynamics of ClpP from Staphylococcus aureus (SaClpP) in wild-type and mutant forms in an effort to discover conformational hotspots that regulate its function. Wild-type SaClpP was found exclusively in the active extended form, with the N-terminal domains of its component protomers in predominantly β-hairpin conformations that are less well-defined than other regions of the protein. A hydrophobic site was identified that, upon mutation, leads to unfolding of the N-terminal domains, loss of SaClpP activity, and formation of a previously unobserved split-ring conformation with a pair of 20-Å-wide pores in the side of the complex. The extended form of the structure and partial activity can be restored via binding of ADEP small-molecule activators. The observed structural plasticity of the N-terminal gates is shown to be a conserved feature through studies of Escherichia coli and Neisseria meningitidis ClpP, suggesting a potential avenue for the development of molecules to allosterically modulate the function of ClpP.

Background Exportin 1 (XPO1/CRM1) is a key mediator of nuclear export with relevance to multiple cancers, including chronic lymphocytic leukemia (CLL). Whole exome sequencing has identified hot-spot somatic XPO1 point mutations which we found to disrupt highly conserved biophysical interactions in the NES-binding groove, conferring novel cargo-binding abilities and forcing cellular mis-localization of critical regulators. However, the pathogenic role played by change-in-function XPO1 mutations in CLL is not fully understood. Methods We performed a large, multi-center retrospective analysis of CLL cases ( N  = 1286) to correlate nonsynonymous mutations in XPO1 (predominantly E571K or E571G; n  = 72) with genetic and epigenetic features contributing to the overall outcomes in these patients. We then established a mouse model with over-expression of wildtype (wt) or mutant (E571K or E571G) XPO1 restricted to the B cell compartment (Eµ-XPO1). Eµ-XPO1 mice were then crossed with the Eµ-TCL1 CLL mouse model. Lastly, we determined crystal structures of XPO1 (wt or E571K) bound to several selective inhibitors of nuclear export (SINE) molecules (KPT-185, KPT-330/Selinexor, and KPT-8602/Eltanexor). Results We report that nonsynonymous mutations in XPO1 associate with high risk genetic and epigenetic features and accelerated CLL progression. Using the newly-generated Eµ-XPO1 mouse model, we found that constitutive B-cell over-expression of wt or mutant XPO1 could affect development of a CLL-like disease in aged mice. Furthermore, concurrent B-cell expression of XPO1 with E571K or E571G mutations and TCL1 accelerated the rate of leukemogenesis relative to that of Eµ-TCL1 mice. Lastly, crystal structures of E571 or E571K-XPO1 bound to SINEs, including Selinexor, are highly similar, suggesting that the activity of this class of compounds will not be affected by XPO1 mutations at E571 in patients with CLL. Conclusions These findings indicate that mutations in XPO1 at E571 can drive leukemogenesis by priming the pre-neoplastic lymphocytes for acquisition of additional genetic and epigenetic abnormalities that collectively result in neoplastic transformation.

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of antituberculosis (anti-TB) therapeutics that target the Clp system. Here, we present a combined biophysical and biochemical study to explore the structure–dynamics–function relationship in MtClpP1P2. Electron cryomicroscopy (cryo-EM) structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key β-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that, on binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod–Wyman–Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function and shows extensive allosteric interactions involving both intra- and interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis.

Despite improvements in the management of chronic hepatitis B (CHB), risk of cirrhosis and hepatocellular carcinoma remains. While hepatitis B surface antigen loss is the optimal end point, safe discontinuation of nucleos(t)ide analog (NA) therapy is controversial because of the possibility of severe or fatal reactivation flares. This is a multicenter cohort study of virally suppressed, end-of-therapy (EOT) hepatitis B e antigen (HBeAg)-negative CHB patients who stopped NA therapy (n = 1,557). Survival analysis techniques were used to analyze off-therapy rates of hepatic decompensation and differences by patient characteristics. We also examined a subgroup of noncirrhotic patients with consolidation therapy of ≥12 months before cessation (n = 1,289). Hepatic decompensation was considered related to therapy cessation if diagnosed off therapy or within 6 months of starting retreatment. Among the total cohort (11.8% diagnosed with cirrhosis, 84.2% start-of-therapy HBeAg-negative), 20 developed hepatic decompensation after NA cessation; 10 events were among the subgroup. The cumulative incidence of hepatic decompensation at 60 months off therapy among the total cohort and subgroup was 1.8% and 1.1%, respectively. The hepatic decompensation rate was higher among patients with cirrhosis (hazard ratio [HR] 5.08, P < 0.001) and start-of-therapy HBeAg-positive patients (HR 5.23, P < 0.001). This association between start-of-therapy HBeAg status and hepatic decompensation remained significant even among the subgroup (HR 10.5, P < 0.001). Patients with cirrhosis and start-of-therapy HBeAg-positive patients should be carefully assessed before stopping NAs to prevent hepatic decompensation. Frequent monitoring of viral and host kinetics after cessation is crucial to determine patient outcome.