Fluorescence-activated droplet sequencing (FAD-seq) directly provides sequences of screening hits in antibody discovery.

Droplet microfluidics has become a very powerful tool in high-throughput screening, including antibody discovery. Screens are usually carried out by physically sorting droplets hosting cells of the desired phenotype, breaking them, recovering the encapsulated cells, and sequencing the paired antibody light and heavy chain genes at the single-cell level. This series of multiple consecutive manipulation steps of rare screening hits is complex and challenging, resulting in a significant loss of clones with the desired phenotype or large fractions of cells with incomplete antibody information. Here, we present fluorescence-activated droplet sequencing, in which droplets showing the desired phenotype are selectively picoinjected with reagents for RT-PCR. Subsequently, light and heavy chain genes are natively paired, fused into a single-chain fragment variant format, and amplified before off-chip transfer and downstream nanopore sequencing. This workflow is sufficiently sensitive for obtaining different paired full-length antibody sequences from as little as five droplets, fulfilling the desired phenotype. Replacing physical sorting by specific sequencing overcomes a general bottleneck in droplet microfluidic screening and should be compatible with many more applications.

The ric-8b protein (resistance to inhibitors of cholinesterase 8b) is key to preserving contractile function in the adult heart.

Resistance to inhibitors of cholinesterases (ric-8 proteins) are involved in modulating G-protein function, but little is known of their potential physiological importance in the heart. In the present study, we assessed the role of resistance to inhibitors of cholinesterase 8b (Ric-8b) in determining cardiac contractile function. We developed a murine model in which it was possible to conditionally delete ric-8b in cardiac tissue in the adult animal after the addition of tamoxifen. Deletion of ric-8b led to severely reduced contractility as measured using echocardiography days after administration of tamoxifen. Histological analysis of the ventricular tissue showed highly variable myocyte size, prominent fibrosis, and an increase in cellular apoptosis. RNA sequencing revealed transcriptional remodeling in response to cardiac ric-8b deletion involving the extracellular matrix and inflammation. Phosphoproteomic analysis revealed substantial downregulation of phosphopeptides related to myosin light chain 2. At the cellular level, the deletion of ric-8b led to loss of activation of the L-type calcium channel through the ß-adrenergic pathways. Using fluorescence resonance energy transfer-based assays, we showed ric-8b protein selectively interacts with the stimulatory G-protein, Gαs. We explored if deletion of Gnas (the gene encoding Gαs) in cardiac tissue using a similar approach in the mouse led to an equivalent phenotype. The conditional deletion of the Gαs gene in the ventricle led to comparable effects on contractile function and cardiac histology. We conclude that ric-8b is essential to preserve cardiac contractile function likely through an interaction with the stimulatory G-protein and downstream phosphorylation of myosin light chain 2.

Mechanisms underlying distinct subcellular localization and regulation of epithelial long myosin light-chain kinase splice variants.

Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn's disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.

Kinesin light chain 1 stabilizes insulin receptor substrate 1 to regulate the IGF-1-AKT signaling pathway during myoblast differentiation.

The IGF signaling pathway plays critical role in regulating skeletal myogenesis. We have demonstrated that KIF5B, the heavy chain of kinesin-1 motor, promotes myoblast differentiation through regulating IGF-p38MAPK activation. However, the roles of the kinesin light chain (Klc) in IGF pathway and myoblast differentiation remain elusive. In this study, we found that Klc1 was upregulated during muscle regeneration and downregulated in senescence mouse muscles and dystrophic muscles from mdx (X-linked muscular dystrophic) mice. Gain- and loss-of-function experiments further displayed that Klc1 promotes AKT-mTOR activity and positively regulates myogenic differentiation. We further identified that the expression levels of IRS1, the critical node of IGF-1 signaling, are downregulated in Klc1-depleted myoblasts. Coimmunoprecipitation study revealed that IRS1 interacted with the 88-154 amino acid sequence of Klc1 via its PTB domain. Notably, the reduced Klc1 levels were found in senescence and osteoporosis skeletal muscle samples from both mice and human. Taken together, our findings suggested a crucial role of Klc1 in the regulation of IGF-AKT pathway during myogenesis through stabilizing IRS1, which might ultimately influence the development of muscle-related disorders.

Cavß3 Contributes to the Maintenance of the Blood-Brain Barrier and Alleviates Symptoms of Experimental Autoimmune Encephalomyelitis.

BACKGROUND: Tight control of cytoplasmic Ca2+ concentration in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavß3, a subunit of voltage-gated Ca2+ (Cav) channels, in modulating Ca2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier. METHODS: We investigated the function of Cavß3 in BMECs by Ca2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavß3-/- (Cavß3-deficient) mice as controls. RESULTS: We identified Cavß3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavß3. After induction of experimental autoimmune encephalomyelitis, Cavß3-/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavß3-/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca2+-dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavß3-/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavß3-/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavß3 with inositol 1,4,5-trisphosphate receptor proteins. CONCLUSIONS: Independent of its function as a subunit of Cav channels, Cavß3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca2+ concentration and Ca2+-dependent MLC phosphorylation in BMECs, and this role of Cavß3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.

Ferritin: Significance in viral infections.

As an indispensable trace element, iron is essential for many biological processes. Increasing evidence has shown that virus infection can perturb iron metabolism and play a role in the occurrence and development of viral infection-related diseases. Ferritin plays a crucial role in maintaining the body's iron homoeostasis. It is an important protein to stabilise the iron balance in cells. Ferritin is a 24-mer hollow iron storage protein composed of two subunits: ferritin heavy chain and ferritin light chain. It was reported that ferritin is not only an intra-cellular iron storage protein, but also a pathogenic mediator that enhances the inflammatory process and stimulates the further inflammatory pathway, which is a key member of the vicious pathogenic cycle to perpetuate. Ferritin exerts immuno-suppressive and pro-inflammatory functions during viral infection. In this review, we describe in detail the basic information of ferritin in the first section, including its structural features, the regulation of ferritin. In the second part, we focus on the role of ferritin in viral infection-related diseases and the molecular mechanisms by which viral infection regulates ferritin. The last section briefly outlines the potential of ferritin in antiviral therapy. Given the importance of iron and viral infection, understanding the role of ferritin during viral infection helps us understand the relationship between iron metabolic dysfunction and viral infection, which provides a new direction for the development of antiviral therapeutic drugs.

Human herpesvirus 6A and axonal injury before the clinical onset of multiple sclerosis.

Recent research indicates that multiple sclerosis is preceded by a prodromal phase with elevated levels of serum neurofilament light chain (sNfL), a marker of axonal injury. The effect of environmental risk factors on the extent of axonal injury during this prodrome is unknown. Human herpesvirus 6A (HHV-6A) is associated with an increased risk of developing multiple sclerosis. The objective of this study was to determine if HHV-6A serostatus is associated with the level of sNfL in the multiple sclerosis prodrome, which would support a causative role of HHV-6A. A nested case-control study was performed by crosslinking multiple sclerosis registries with Swedish biobanks. Individuals with biobank samples collected before the clinical onset of multiple sclerosis were included as cases. Controls without multiple sclerosis were randomly selected, matched for biobank, sex, sampling date and age. Serostatus of HHV-6A and Epstein-Barr virus was analysed with a bead-based multiplex assay. The concentration of sNfL was analysed with single molecule array technology. The association between HHV-6A serology and sNfL was assessed by stratified t-tests and linear regressions, adjusted for Epstein-Barr virus serostatus and sampling age. Within-pair ratios of HHV-6A seroreactivity and sNfL were calculated for each case and its matched control. To assess the temporal relationship between HHV-6A antibodies and sNfL, these ratios were plotted against the time to the clinical onset of multiple sclerosis and compared using locally estimated scatterplot smoothing regressions with 95% confidence intervals (CI). Samples from 519 matched case-control pairs were included. In cases, seropositivity of HHV-6A was significantly associated with the level of sNfL (+11%, 95% CI 0.2-24%, P = 0.045) and most pronounced in the younger half of the cases (+24%, 95% CI 6-45%, P = 0.007). No such associations were observed among the controls. Increasing seroreactivity against HHV-6A was detectable before the rise of sNfL (significant within-pair ratios from 13.6 years versus 6.6 years before the clinical onset of multiple sclerosis). In this study, we describe the association between HHV-6A antibodies and the degree of axonal injury in the multiple sclerosis prodrome. The findings indicate that elevated HHV-6A antibodies both precede and are associated with a higher degree of axonal injury, supporting the hypothesis that HHV-6A infection may contribute to multiple sclerosis development in a proportion of cases.

Leriglitazone halts disease progression in adult patients with early cerebral adrenoleukodystrophy.

Cerebral adrenoleukodystrophy (CALD) is an X-linked rapidly progressive demyelinating disease leading to death usually within a few years. The standard of care is haematopoietic stem cell transplantation (HSCT), but many men are not eligible due to age, absence of a matched donor or lesions of the corticospinal tracts (CST). Based on the ADVANCE study showing that leriglitazone decreases the occurrence of CALD, we treated 13 adult CALD patients (19-67 years of age) either not eligible for HSCT (n = 8) or awaiting HSCT (n = 5). Patients were monitored every 3 months with standardized neurological scores, plasma biomarkers and brain MRI comprising lesion volumetrics and diffusion tensor imaging. The disease stabilized clinically and radiologically in 10 patients with up to 2 years of follow-up. Five patients presented with gadolinium enhancing CST lesions that all turned gadolinium negative and, remarkably, regressed in four patients. Plasma neurofilament light chain levels stabilized in all 10 patients and correlated with lesion load. The two patients who continued to deteriorate were over 60 years of age with prominent cognitive impairment. One patient died rapidly from coronavirus disease 2019. These results suggest that leriglitazone can arrest disease progression in adults with early-stage CALD and may be an alternative treatment to HSCT.

Serum and CSF biomarkers in asymptomatic patients during primary HIV infection: a randomized study.

It is debated whether central nervous system involvement begins during acute HIV infection in persons without meningitis/encephalitis and if specific antiretroviral drugs or combinations would be beneficial. Neurologically asymptomatic participants enrolled in a randomized and controlled study comparing three combination antiretroviral regimens (tenofovir alafenamide/emtricitabine plus dolutegravir, darunavir or both) during primary HIV infection were enrolled. Serum and cerebrospinal fluid (CSF) were collected at baseline, 12 and 48 (serum only) weeks after treatment initiation. Single Molecule Array was used to measure neurofilament light chain (NFL), total tau protein (Tau), Brain-Derived Neurotrophic Factor (BDNF), Glial Fibrillary Acidic Protein (GFAP), Ubiquitin C-terminal Hydrolase (UCH-L1). We assessed the longitudinal change in biomarkers over time as well as the change in the prevalence of serum NFL concentrations above previously published age-adjusted cut-offs (7 pg/mL if 5-18 years, 10 pg/mL if 18-51 years, 15 pg/mL if 51-61 years, 20 pg/mL if 61-70 years and 35 pg/mL if >70 years). Serum was available from 47 participants at all time points while CSF was in 13 and 7 participants (baseline/W12). We observed a significant direct serum-to-CSF correlation for NFL (rho = 0.692, p = 0.009), GFAP (rho = 0.659, p = 0.014) and BDNF (rho = 0.587, p = 0.045). Serum (rho = 0.560, p = 0.046) and CSF NFL (rho = 0.582, p = 0.037) concentrations were directly associated with CSF HIV RNA levels. We observed a significant decrease over time in serum NFL (p = 0.006) and GFAP (p = 0.006) but not in the other biomarkers. No significant difference was observed among the treatment arms. At baseline, serum and CSF age-adjusted NFL levels were above age-adjusted cut-offs in 23 (48.9%) and 4 participants (30.8%); considering serum NFL, this proportion was lower at weeks 12 (31.9%, p = 0.057) and 48 (27.7%, p = 0.13). A relevant proportion of neurologically asymptomatic participants had abnormal CSF and serum NFL levels during primary HIV infection. NFL and GFAP decreased in serum following combination antiretroviral therapy without significant differences among the treatment arms.

Serum biomarkers at disease onset for personalized therapy in multiple sclerosis.

The potential of combining serum neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) levels to predict disability worsening in multiple sclerosis (MS) remains underexplored. We aimed to investigate whether sNfL and sGFAP values identify distinct subgroups of patients according to the risk of disability worsening and their response to disease-modifying treatments (DMTs). This multicentre study, conducted across thirteen European hospitals, spanned from July 15, 1994, to August 18, 2022, with follow-up until September 26, 2023. We enrolled MS patients who had serum samples collected within 12 months from disease onset and before initiating DMTs. Multivariable regression models were used to estimate the risk of relapse-associated worsening (RAW), progression independent of relapse activity (PIRA), and Expanded Disability Status Scale (EDSS) score of 3. Of the 725 patients included, median age was 34.2 years (IQR, 27.6-42.4), and 509 patients (70.2%) were female. Median follow-up duration was 6.43 years (IQR, 4.65-9.81). Higher sNfL values associated with an elevated risk of RAW (HR of 1.45; 95% CI 1.19-1.76; P < 0.001), PIRA (HR of 1.43; 95% CI 1.13-1.81; P = 0.003), and reaching an EDSS of 3 (HR of 1.55; 95% CI 1.29-1.85; P < 0.001). Moreover, higher sGFAP levels were linked to a higher risk of achieving an EDSS score of 3 (HR of 1.36; 95% CI 1.06-1.74; P = 0.02) and, in patients with low sNfL values, to PIRA (HR of 1.86; 95% CI 1.01-3.45; P = 0.04). We further examined the combined effect of sNfL and sGFAP levels. Patients with low sNfL and sGFAP values (NLGL) exhibited a low risk of all outcomes and served as reference. Untreated patients with high sNfL levels showed a higher risk of RAW, PIRA, and reaching an EDSS of 3. Injectable or oral DMTs reduced the risk of RAW in these patients but failed to mitigate the risk of PIRA and reaching an EDSS of 3. Conversely, high-efficacy DMTs counteracted the heightened risk of these outcomes, except for the risk of PIRA in patients with high sNfL and sGFAP levels. Patients with low sNfL and high sGFAP values (NLGH) showed an increased risk of PIRA and achieving an EDSS of 3, which remained unchanged with either high-efficacy or other DMTs. In conclusion, evaluating sNfL and sGFAP levels at disease onset in MS may identify distinct phenotypes associated with diverse immunological pathways of disability acquisition and therapeutic response.

Uncovering neural substrates across Alzheimer's disease stages using contrastive variational autoencoder.

Alzheimer's disease is the most common major neurocognitive disorder. Although currently, no cure exists, understanding the neurobiological substrate underlying Alzheimer's disease progression will facilitate early diagnosis and treatment, slow disease progression, and improve prognosis. In this study, we aimed to understand the morphological changes underlying Alzheimer's disease progression using structural magnetic resonance imaging data from cognitively normal individuals, individuals with mild cognitive impairment, and Alzheimer's disease via a contrastive variational autoencoder model. We used contrastive variational autoencoder to generate synthetic data to boost the downstream classification performance. Due to the ability to parse out the nonclinical factors such as age and gender, contrastive variational autoencoder facilitated a purer comparison between different Alzheimer's disease stages to identify the pathological changes specific to Alzheimer's disease progression. We showed that brain morphological changes across Alzheimer's disease stages were significantly associated with individuals' neurofilament light chain concentration, a potential biomarker for Alzheimer's disease, highlighting the biological plausibility of our results.

Neurofilament Light Chain as a Biomarker of Neuronal Damage in Children With Malaria.

Malaria can cause brain injury. Neurofilament light chain (NfL) is a biomarker of neuronal damage. Here we examined longitudinal plasma NfL levels in children aged 1-12 years with uncomplicated and severe malaria from Mozambique. NfL levels were similar in all malaria cases at hospital admission. However, levels increased over time and the increment was significantly higher in severe malaria cases with neurological manifestations (ie, coma, impaired consciousness, or repeated seizures). NfL may be useful to identify and quantify brain injury in malaria.

Drebrin is Required for Myosin-facilitated Actin Cytoskeletal Remodeling during Pulmonary Alveolar Development.

Alveolar septation increases gas-exchange surface area and requires coordinated cytoskeletal rearrangement in lung fibroblasts (LFs) to balance the demands of contraction and cell migration. We hypothesized that DBN (drebrin), a modulator of the actin cytoskeleton in neuronal dendrites, regulates the remodeling of the LF cytoskeleton. Using mice bearing a transgelin-Cre-targeted deletion of Dbn in pulmonary fibroblasts and pericytes, we examined alterations in alveolar septal outgrowth, LF spreading and migration, and actomyosin function. The alveolar surface area and number of alveoli were reduced, whereas alveolar ducts were enlarged, in mice bearing the dbn deletion (DBNΔ) compared with their littermates bearing only one dbn-Flox allele (control). Cultured DBNΔ LFs were deficient in their responses to substrate rigidity and migrated more slowly. Drebrin was abundant in the actin cortex and lamella, and the actin fiber orientation was less uniform in lamella of DBNΔ LFs, which limited the development of traction forces and altered focal adhesion dynamics. Actin fiber orientation is regulated by contractile NM2 (nonmuscle myosin-2) motors, which help arrange actin stress fibers into thick ventral actin stress fibers. Using fluorescence anisotropy, we observed regional intracellular differences in myosin regulatory light chain phosphorylation in control LFs that were altered by dbn deletion. Using perturbations to induce and then release stalling of NM2 on actin in LFs from both genotypes, we made predictions explaining how DBN interacts with actin and NM2. These studies provide new insight for diseases such as emphysema and pulmonary fibrosis, in which fibroblasts inappropriately respond to mechanical cues in their environment.

Ensembles of human myosin-19 bound to calmodulin and regulatory light chain RLC12B drive multimicron transport.

Myosin-19 (Myo19) controls the size, morphology, and distribution of mitochondria, but the underlying role of Myo19 motor activity is unknown. Complicating mechanistic in vitro studies, the identity of the light chains (LCs) of Myo19 remains unsettled. Here, we show by coimmunoprecipitation, reconstitution, and proteomics that the three IQ motifs of human Myo19 expressed in Expi293 human cells bind regulatory light chain (RLC12B) and calmodulin (CaM). We demonstrate that overexpression of Myo19 in HeLa cells enhances the recruitment of both Myo19 and RLC12B to mitochondria, suggesting cellular association of RLC12B with the motor. Further experiments revealed that RLC12B binds IQ2 and is flanked by two CaM molecules. In vitro, we observed that the maximal speed (∼350 nm/s) occurs when Myo19 is supplemented with CaM, but not RLC12B, suggesting maximal motility requires binding of CaM to IQ-1 and IQ-3. The addition of calcium slowed actin gliding (∼200 nm/s) without an apparent effect on CaM affinity. Furthermore, we show that small ensembles of Myo19 motors attached to quantum dots can undergo processive runs over several microns, and that calcium reduces the attachment frequency and run length of Myo19. Together, our data are consistent with a model where a few single-headed Myo19 molecules attached to a mitochondrion can sustain prolonged motile associations with actin in a CaM- and calcium-dependent manner. Based on these properties, we propose that Myo19 can function in mitochondria transport along actin filaments, tension generation on multiple randomly oriented filaments, and/or pushing against branched actin networks assembled near the membrane surface.

Computational evidences of a misfolding event in an aggregation-prone light chain preceding the formation of the non-native pathogenic dimer.

Antibody light chain amyloidosis is a disorder in which protein aggregates, mainly composed of immunoglobulin light chains, deposit in diverse tissues impairing the correct functioning of organs. Interestingly, due to the high susceptibility of antibodies to mutations, AL amyloidosis appears to be strongly patient-specific. Indeed, every patient will display their own mutations that will make the proteins involved prone to aggregation thus hindering the study of this disease on a wide scale. In this framework, determining the molecular mechanisms that drive the aggregation could pave the way to the development of patient-specific therapeutics. Here, we focus on a particular patient-derived light chain, which has been experimentally characterized. We investigated the early phases of the aggregation pathway through extensive full-atom molecular dynamics simulations, highlighting a structural rearrangement and the exposure of two hydrophobic regions in the aggregation-prone species. Next, we moved to consider the pathological dimerization process through docking and molecular dynamics simulations, proposing a dimeric structure as a candidate pathological first assembly. Overall, our results shed light on the first phases of the aggregation pathway for a light chain at an atomic level detail, offering new structural insights into the corresponding aggregation process.

Restoration of Cardiac Myosin Light Chain Kinase Ameliorates Systolic Dysfunction by Reducing Superrelaxed Myosin.

BACKGROUND: Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3, regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure. METHODS: We generated the knock-in mice (Mylk3+/fs and Mylk3fs/fs) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation (MYLK3+/fs) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154). RESULTS: Both mice (Mylk3+/fs and Mylk3fs/fs) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the Vmax for ventricular myosin regulatory light chain phosphorylation without affecting the Km. LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3/PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes. CONCLUSIONS: cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.

Role of ZIP kinase in development of myofibroblast differentiation from HPMCs.

During the development of pleural fibrosis, pleural mesothelial cells (PMCs) undergo phenotypic switching from differentiated mesothelial cells to mesenchymal cells (MesoMT). Here, we investigated how external stimuli such as TGF-ß induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-ß significantly increased di-phosphorylation but not mono-phosphorylation of myosin II regulatory light chain (RLC) in HPMCs. An increase in RLC di-phosphorylation was also found at the pleural layer of our carbon black bleomycin (CBB) pleural fibrosis mouse model, where it showed filamentous localization that coincided with alpha smooth muscle actin (αSMA) in the cells in the pleura. Among the protein kinases that can phosphorylate myosin II RLC, ZIPK (zipper-interacting kinase) protein expression was significantly augmented after TGF-ß stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-ß significantly increased the expression of ZIP kinase protein, the change in ZIP kinase mRNA was marginal, suggesting a posttranscriptional mechanism for the regulation of ZIP kinase expression by TGF-ß. ZIPK gene knockdown (KD) also significantly reduced TGF-ß-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-ß-induced contractility of HPMCs consistent with siZIPK-induced decrease in the di-phosphorylation of myosin II RLC. The present results implicate ZIPK in the regulation of the contractility of HPMC-derived myofibroblasts, phenotype switching, and myofibroblast differentiation of HPMCs.NEW & NOTEWORTHY Here, we highlight that ZIP kinase is responsible for di-phosphorylation of myosin light chain, which facilitates stress fiber formation and actomyosin-based cell contraction during mesothelial to mesenchymal transition in human pleural mesothelial cells. This transition has a significant impact on tissue remodeling and subsequent stiffness of the pleura. This study provides insight into a new therapeutic strategy for the treatment of pleural fibrosis.

Cognitively healthy APOE4/4 carriers show white matter impairment associated with serum NfL and amyloid-PET.

Except for aging, carrying the APOE ε4 allele (APOE4) is the most important risk factor for sporadic Alzheimer's disease. APOE4 carriers may have reduced capacity to recycle lipids, resulting in white matter microstructural abnormalities. In this study, we evaluated whether white matter impairment measured by diffusion tensor imaging (DTI) differs between healthy individuals with a different number of APOE4 alleles, and whether white matter impairment associates with brain beta-amyloid (Aß) load and serum levels of neurofilament light chain (NfL). We studied 96 participants (APOE3/3, N = 37; APOE3/4, N = 39; APOE4/4, N = 20; mean age 70.7 (SD 5.22) years, 63% females) with a brain MRI including a DTI sequence (N = 96), Aß-PET (N = 89) and a venous blood sample for the serum NfL concentration measurement (N = 88). Fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (RD) and axial diffusivity (AxD) in six a priori-selected white matter regions-of-interest (ROIs) were compared between the groups using ANCOVA, with sex and age as covariates. A voxel-weighted average of FA, MD, RD and AxD was calculated for each subject, and correlations with Aß-PET and NfL levels were evaluated. APOE4/4 carriers exhibited a higher MD and a higher RD in the body of corpus callosum than APOE3/4 (p = 0.0053 and p = 0.0049, respectively) and APOE3/3 (p = 0.026 and p = 0.042). APOE4/4 carriers had a higher AxD than APOE3/4 (p = 0.012) and APOE3/3 (p = 0.040) in the right cingulum adjacent to cingulate cortex. In the total sample, composite MD, RD and AxD positively correlated with the cortical Aß load (r = 0.26 to 0.33, p < 0.013 for all) and with serum NfL concentrations (r = 0.31 to 0.36, p < 0.0028 for all). In conclusion, increased local diffusivity was detected in cognitively unimpaired APOE4/4 homozygotes compared to APOE3/4 and APOE3/3 carriers, and increased diffusivity correlated with biomarkers of Alzheimer's disease and neurodegeneration. White matter impairment seems to be an early phenomenon in the Alzheimer's disease pathologic process in APOE4/4 homozygotes.

Monoclonal immunoglobulin crystalline nephropathies.

Monoclonal Ig crystalline nephropathies are rare lesions resulting from precipitation of monoclonal Igs in the kidney as crystalline inclusions. They can be categorized into lesions with predominant intracellular crystals (light chain [LC] proximal tubulopathy, LC crystal-storing histiocytosis, and LC crystalline podocytopathy) and lesions with predominant extracellular crystals (crystalglobulin-induced nephropathy and crystalline variant of LC cast nephropathy). The majority of these lesions are associated with low tumor burden lymphoproliferative disorders, with the exception of crystalline variant of LC cast nephropathy. Extrarenal involvement (e.g., skin and cornea) is frequent. Kidney biopsy is the cornerstone for diagnosis, which often requires electron microscopy and antigen retrieval. A thorough hematologic workup and evaluation of extrarenal involvement is mandatory for management. Treatment of these lesions is with clone-directed therapy, with the goal of achieving hematologic very good partial response or complete response, which preserves or improves kidney function. In vitro and in vivo studies, animal models, and novel sequencing techniques have been invaluable tools to understand the pathogenesis of LC proximal tubulopathy and can be used to increase our limited knowledge of the pathogenesis of the other monoclonal Ig crystalline nephropathies. This review provides an update on the pathology, renal and hematologic characteristics, extrarenal manifestations, prognosis, treatment, and pathogenesis of monoclonal Ig crystalline nephropathies.

Methylation of Interleukin-1 receptor-associated kinase-3 and the risk of multiple sclerosis relapse/activity.

This study retrospectively investigated the impact of interleukin-1 receptor-associated kinase-3 (IRAK-3/IRAK-M) silencing by methylation on the likelihood of multiple sclerosis (MS) activity. This cross-sectional study included 90 patients with MS: 45 with active disease (Group 1), 45 in remission (Group 2), and 45 healthy controls. The study included quantitation of IRAK-3 methylation index (MI%), IRAK-3 mRNA, and myeloid differentiation factor88 (MyD88) and assessment of NF-κB activity. IRAK-3 MI% was significantly higher in group 1 compared to group 2, accompanied by lower IRAK-3 mRNA expression, elevated circulating MyD88, and increased NF-κB activity. IRAK-3 MI% correlated negatively with its transcript and positively with MyD88 and NF-κB activity. A logistic regression model was created to predict active demyelination. The C-index was 0.924, which indicates a very strong prediction model. Within the limitations of current work, IRAK-3 methylation level seems to be a promising candidate biomarker for identifying MS patients at risk of relapse.