Pro-Brain-Derived Neurotrophic Factor (BDNF), but Not Mature BDNF, Is Expressed in Human Skeletal Muscle: Implications for Exercise-Induced Neuroplasticity.

Exercise promotes brain plasticity partly by stimulating increases in mature brain-derived neurotrophic factor (mBDNF), but the role of the pro-BDNF isoform in the regulation of BDNF metabolism in humans is unknown. We quantified the expression of pro-BDNF and mBDNF in human skeletal muscle and plasma at rest, after acute exercise (+/- lactate infusion), and after fasting. Pro-BDNF and mBDNF were analyzed with immunoblotting, enzyme-linked immunosorbent assay, immunohistochemistry, and quantitative polymerase chain reaction. Pro-BDNF was consistently and clearly detected in skeletal muscle (40-250 pg mg-1 dry muscle), whereas mBDNF was not. All methods showed a 4-fold greater pro-BDNF expression in type I muscle fibers compared to type II fibers. Exercise resulted in elevated plasma levels of mBDNF (55%) and pro-BDNF (20%), as well as muscle levels of pro-BDNF (∼10%, all P < 0.05). Lactate infusion during exercise induced a significantly greater increase in plasma mBDNF (115%, P < 0.05) compared to control (saline infusion), with no effect on pro-BDNF levels in plasma or muscle. A 3-day fast resulted in a small increase in plasma pro-BDNF (∼10%, P < 0.05), with no effect on mBDNF. Pro-BDNF is highly expressed in human skeletal muscle, particularly in type I fibers, and is increased after exercise. While exercising with higher lactate augmented levels of plasma mBDNF, exercise-mediated increases in circulating mBDNF likely derive partly from release and cleavage of pro-BDNF from skeletal muscle, and partly from neural and other tissues. These findings have implications for preclinical and clinical work related to a wide range of neurological disorders such as Alzheimer's, clinical depression, and amyotrophic lateral sclerosis.

Mutation mapping of a variegated EMS tomato reveals an FtsH-like protein precursor potentially causing patches of four phenotype classes in the leaves with distinctive internal morphology.

BACKGROUND: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F2 population to reveal candidate genes responsible for the variegation. RESULTS: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation. CONCLUSION: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation.

Pre-proenkephalin 1 is Downregulated Under Unloading and is Involved in Osteoblast Biology.

Pre-proenkephalin 1 (Penk1) is a pro-neuropeptide that belongs to the typical opioid peptide's family, having analgesic properties. We previously found Penk1 to be the most downregulated gene in a whole gene profiling analysis performed in osteoblasts subjected to microgravity as a model of mechanical unloading. In this work, Penk1 downregulation was confirmed in the bones of two in vivo models of mechanical unloading: tail-suspended and botulinum toxin A (botox)-injected mice. Consistently, in the sera from healthy volunteers subjected to bed rest, we observed an inverse correlation between PENK1 and bed rest duration. These results prompted us to investigate a role for this factor in bone. Penk1 was highly expressed in mouse bone, but its global deletion failed to impact bone metabolism in vivo. Indeed, Penk1 knock out (Penk1-/-) mice did not show an overt bone phenotype compared to the WT littermates. Conversely, in vitro Penk1 gene expression progressively increased during osteoblast differentiation and its transient silencing in mature osteoblasts by siRNAs upregulated the transcription of the Sost1 gene encoding sclerostin, and decreased Wnt3a and Col1a1 mRNAs, suggesting an altered osteoblast activity due to an impairment of the Wnt pathway. In line with this, osteoblasts treated with the Penk1 encoded peptide, Met-enkephalin, showed an increase of Osx and Col1a1 mRNAs and enhanced nodule mineralization. Interestingly, primary osteoblasts isolated from Penk1-/- mice showed lower metabolic activity, ALP activity, and nodule mineralization, as well as a lower number of CFU-F compared to osteoblasts isolated from WT mice, suggesting that, unlike the transient inhibition, the chronic Penk1 deletion affects both osteoblast differentiation and activity. Taken together, these results highlight a role for Penk1 in the regulation of the response of the bone to mechanical unloading, potentially acting on osteoblast differentiation and activity in a cell-autonomous manner.

Loss of Preproinsulin Interaction with Signal Recognition Particle Activates Protein Quality Control, Decreasing mRNA Stability.

Many insulin gene variants alter the protein sequence and result in monogenic diabetes due to insulin insufficiency. However, the molecular mechanisms of various disease-causing mutations are unknown. Insulin is synthesized as preproinsulin containing a signal peptide (SP). SPs of secreted proteins are recognized by the signal recognition particle (SRP) or by another factor in a SRP-independent pathway. If preproinsulin uses SRP-dependent or independent pathways is still debatable. We demonstrate by the use of site-specific photocrosslinking that the SRP subunit, SRP54, interacts with the preproinsulin SP. Moreover, SRP54 depletion leads to the decrease of insulin mRNA and protein expression, supporting the involvement of the RAPP protein quality control in insulin biogenesis. RAPP regulates the quality of secretory proteins through degradation of their mRNA. We tested five disease-causing mutations in the preproinsulin SP on recognition by SRP and on their effects on mRNA and protein levels. We demonstrate that the effects of mutations are associated with their position in the SP and their severity. The data support diverse molecular mechanisms involved in the pathogenesis of these mutations. We show for the first time the involvement of the RAPP protein quality control pathway in insulin biogenesis that is implicated in the development of neonatal diabetes caused by the Leu13Arg mutation.

Effect of prothymosin α on neuroplasticity following cerebral ischemia­reperfusion injury.

Prothymosin α (ProT), a highly acidic nuclear protein with multiple cellular functions, has shown potential neuroprotective properties attributed to its anti­necrotic and anti­apoptotic activities. The present study aimed to investigate the beneficial effect of ProT on neuroplasticity after ischemia­reperfusion injury and elucidate its underlying mechanism of action. Primary cortical neurons were either treated with ProT or overexpressing ProT by gene transfection and exposed to oxygen­glucose deprivation for 2 h in vitro. Immunofluorescence staining for ProT and MAP­2 was performed to quantify ProT protein expression and assess neuronal arborization. Mice treated with vehicle or ProT (100 µg/kg) and ProT overexpression in transgenic mice received middle cerebral artery occlusion for 50 min to evaluate the effect of ProT on neuroplasticity­associated protein following ischemia­reperfusion injury. The results demonstrated that in cultured neurons ProT significantly increased neurite lengths and the number of branches, accompanied by an upregulation mRNA level of brain­derived neurotrophic factor. Furthermore, ProT administration improved the protein expressions of synaptosomal­associated protein, 25 kDa and postsynaptic density protein 95 after ischemic­reperfusion injury in vivo. These findings suggested that ProT can potentially induce neuroplasticity effects following ischemia­reperfusion injury.

Cysteine-free cone snail venom peptides: Classification of precursor proteins and identification of mature peptides.

The cysteine-free acyclic peptides present in marine cone snail venom have been much less investigated than their disulfide bonded counterparts. Precursor protein sequences derived from transcriptomic data, together with mass spectrometric fragmentation patterns for peptides present in venom duct tissue extracts, permit the identification of mature peptides. Twelve distinct gene superfamiles have been identified with precursor lengths between 64 and 158 residues. In the case of Conus monile, three distinct mature peptides have been identified, arising from two distinct protein precursors. Mature acyclic peptides are often post-translationally modified, with C-terminus amidation, a feature characteristic of neuropeptides. In the present study, 20 acyclic peptides from Conus monile and Conus betulinus were identified. The common modifications of C-terminus amidation, gamma carboxylation of glutamic acid (E to ϒ), N-terminus conversion of Gln (Q) to a pyroglutamyl residue (Z), and hydroxylation of Pro (P) to Hyp (O) are observed in one or more peptides identified in this study. Proteolytic trimming of sequences by cleavage at the C-terminus of Asn (N) residues is established. The presence of an asparagine endopeptidase is strengthened by the identification of legumain-like sequences in the transcriptome assemblies from diverse Conus species. Such sequences may be expected to have a cleavage specificity at Asn-Xxx peptide bonds.

Structural insights into cytokine cleavage by inflammatory caspase-4.

Inflammatory caspases are key enzymes in mammalian innate immunity that control the processing and release of interleukin-1 (IL-1)-family cytokines1,2. Despite the biological importance, the structural basis for inflammatory caspase-mediated cytokine processing has remained unclear. To date, catalytic cleavage of IL-1-family members, including pro-IL-1ß and pro-IL-18, has been attributed primarily to caspase-1 activities within canonical inflammasomes3. Here we demonstrate that the lipopolysaccharide receptor caspase-4 from humans and other mammalian species (except rodents) can cleave pro-IL-18 with an efficiency similar to pro-IL-1ß and pro-IL-18 cleavage by the prototypical IL-1-converting enzyme caspase-1. This ability of caspase-4 to cleave pro-IL-18, combined with its previously defined ability to cleave and activate the lytic pore-forming protein gasdermin D (GSDMD)4,5, enables human cells to bypass the need for canonical inflammasomes and caspase-1 for IL-18 release. The structure of the caspase-4-pro-IL-18 complex determined using cryogenic electron microscopy reveals that pro-lL-18 interacts with caspase-4 through two distinct interfaces: a protease exosite and an interface at the caspase-4 active site involving residues in the pro-domain of pro-IL-18, including the tetrapeptide caspase-recognition sequence6. The mechanisms revealed for cytokine substrate capture and cleavage differ from those observed for the caspase substrate GSDMD7,8. These findings provide a structural framework for the discussion of caspase activities in health and disease.

Invasive mussels fashion silk-like byssus via mechanical processing of massive horizontally acquired coiled coils.

Zebra and quagga mussels (Dreissena spp.) are invasive freshwater biofoulers that perpetrate devastating economic and ecological impact. Their success depends on their ability to anchor onto substrates with protein-based fibers known as byssal threads. Yet, compared to other mussel lineages, little is understood about the proteins comprising their fibers or their evolutionary history. Here, we investigated the hierarchical protein structure of Dreissenid byssal threads and the process by which they are fabricated. Unique among bivalves, we found that threads possess a predominantly ß-sheet crystalline structure reminiscent of spider silk. Further analysis revealed unexpectedly that the Dreissenid thread protein precursors are mechanoresponsive α-helical proteins that are mechanically processed into ß-crystallites during thread formation. Proteomic analysis of the byssus secretory organ and byssus fibers revealed a family of ultrahigh molecular weight (354 to 467 kDa) asparagine-rich (19 to 20%) protein precursors predicted to form α-helical coiled coils. Moreover, several independent lines of evidence indicate that the ancestral predecessor of these proteins was likely acquired via horizontal gene transfer. This chance evolutionary event that transpired at least 12 Mya has endowed Dreissenids with a distinctive and effective fiber formation mechanism, contributing significantly to their success as invasive species and possibly, inspiring new materials design.

[Preparation and Application of Monoclonal Antibody Against Human von Willebrand Factor Propeptide].

OBJECTIVE: To develop monoclonal antibodies that can specifically recognize human von Willebrand factor (VWF) propeptide (VWFpp) in plasma, and establish a rapid and reliable method for the detection of VWFpp antigen in plasma by using the double-antibody sandwich ELISA with the obtained anti-VWFpp monoclonal antibody. METHODS: The recombinant human VWFpp (D1 and D2 regions) protein expressed in eukaryotic cells was used as immunogen to immunize BALB/c mice with routine method, so as to obtain clones of fusion cells. After screening and identification, hybridoma cell lines secreting monoclonal antibodies against VWFpp were selected, and then double-antibody sandwich ELISA assay was used to construct VWFpp antigen detection kit for the determination of VWFpp in human plasma. The levels of VWFpp antigen in plasma of 12 leukemia patients who underwent bone marrow transplantation were dynamically detected. RESULTS: Two hybridoma cell lines that can be subcultured continuously and secrete monoclonal antibodies against VWFpp were obtained and named SZ175 and SZ176 respectively. Identified by ELISA and Western blot, the antibodies could both specifically recognize VWFpp but couldn't recognize mature VWF (without propeptide). Based on the principle of double-antibody sandwich ELISA, monoclonal antibodies SZ175 and SZ176 were successfully made into a kit for detecting VWFpp antigen. The plasma VWFpp levels of leukemia patients before and after bone marrow transplantation were dynamically detected. The results showed that the plasma VWFpp levels of the patients after transplantation were significantly higher than those before transplantation. CONCLUSION: Two monoclonal antibodies against VWFpp were successfully prepared, and a double-antibody sandwich ELISA detection kit for VWFpp antigen was constructed, which provides a powerful tool for further study on the biological function of VWFpp, the clinical diagnosis and classification of von Willebrand disease (VWD), and the prognostic monitoring of endothelial injury-related diseases.

Zonulin as a Potential Therapeutic Target in Microbiota-Gut-Brain Axis Disorders: Encouraging Results and Emerging Questions.

The relationship between dysbiosis and central nervous diseases has been proved in the last 10 years. Microbial alterations cause increased intestinal permeability, and the penetration of bacterial fragment and toxins induces local and systemic inflammatory processes, affecting distant organs, including the brain. Therefore, the integrity of the intestinal epithelial barrier plays a central role in the microbiota-gut-brain axis. In this review, we discuss recent findings on zonulin, an important tight junction regulator of intestinal epithelial cells, which is assumed to play a key role in maintaining of the blood-brain barrier function. In addition to focusing on the effect of microbiome on intestinal zonulin release, we also summarize potential pharmaceutical approaches to modulate zonulin-associated pathways with larazotide acetate and other zonulin receptor agonists or antagonists. The present review also addresses the emerging issues, including the use of misleading nomenclature or the unsolved questions about the exact protein sequence of zonulin.

A Novel Peptide Reagent for Investigating Disulfide-Coupled Folding Intermediates of Mid-Size Proteins.

Investigations of protein folding have largely involved the use of disulfide-containing proteins, since the disulfide-coupled folding of proteins allows folding intermediates to be trapped and their conformations determined. However, studies of the folding mechanisms of mid-size proteins face several problems, one of which is that detecting folding intermediates is difficult. Therefore, to solve this issue, a novel peptide reagent, maleimidohexanoyl-Arg5-Tyr-NH2, was designed and applied to the detection of folding intermediates of model proteins. BPTI was chosen as a model small protein to estimate the ability of the novel reagent to detect folding intermediates. In addition, a precursor protein (prococoonase) of Bombyx mori cocoonase was used as a model mid-size protein. Cocoonase is classified as a serine protease and has a high homology with trypsin. We recently found that the propeptide sequence of prococoonase (proCCN) is important for the folding of cocoonase. However, it was difficult to study the folding pathway of proCCN since the folding intermediates could not be separated on a reversed-phase HPLC (RP-HPLC). Therefore, to separate the folding intermediates by RP-HPLC, the novel labeling reagent was used to accomplish this for proCCN. The results indicated that the peptide reagent allowed the intermediates to be captured, separated on SDS-PAGE, and analyzed by RP-HPLC without the occurrence of undesirable disulfide-exchange reactions during the labeling reactions. The peptide reagent reported herein is a practical tool for investigating the mechanisms of disulfide-coupled folding of mid-size proteins.

A quantitative fluorescence-based approach to study mitochondrial protein import.

Mitochondria play central roles in cellular energy production and metabolism. Most proteins required to carry out these functions are synthesized in the cytosol and imported into mitochondria. A growing number of metabolic disorders arising from mitochondrial dysfunction can be traced to errors in mitochondrial protein import. The mechanisms underlying the import of precursor proteins are commonly studied using radioactively labeled precursor proteins imported into purified mitochondria. Here, we establish a fluorescence-based import assay to analyze protein import into mitochondria. We show that fluorescently labeled precursors enable import analysis with similar sensitivity to those using radioactive precursors, yet they provide the advantage of quantifying import with picomole resolution. We adapted the import assay to a 96-well plate format allowing for fast analysis in a screening-compatible format. Moreover, we show that fluorescently labeled precursors can be used to monitor the assembly of the F1 F0 ATP synthase in purified mitochondria. Thus, we provide a sensitive fluorescence-based import assay that enables quantitative and fast import analysis.

Mitochondrial protein transport: Versatility of translocases and mechanisms.

Biogenesis of mitochondria requires the import of approximately 1,000 different precursor proteins into and across the mitochondrial membranes. Mitochondria exhibit a wide variety of mechanisms and machineries for the translocation and sorting of precursor proteins. Five major import pathways that transport proteins to their functional intramitochondrial destination have been elucidated; these pathways range from the classical amino-terminal presequence-directed pathway to pathways using internal or even carboxy-terminal targeting signals in the precursors. Recent studies have provided important insights into the structural organization of membrane-embedded preprotein translocases of mitochondria. A comparison of the different translocases reveals the existence of at least three fundamentally different mechanisms: two-pore-translocase, ß-barrel switching, and transport cavities open to the lipid bilayer. In addition, translocases are physically engaged in dynamic interactions with respiratory chain complexes, metabolite transporters, quality control factors, and machineries controlling membrane morphology. Thus, mitochondrial preprotein translocases are integrated into multi-functional networks of mitochondrial and cellular machineries.

Accumulated precursors of specific GPI-anchored proteins upregulate GPI biosynthesis with ARV1.

We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is upregulated when endoplasmic reticulum-associated degradation (ERAD) is defective; however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR-Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 are involved in upregulation of GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, with the remaining uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 31 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55, CD48, and PLET1 enhance GPI biosynthesis. ARV1 is prerequisite for the GPI upregulation by CD55 precursor. Our data indicate that GPI biosynthesis is balanced to need by ARV1 and precursors of specific GPI-anchored proteins.

Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord.

Corticospinal neurons (CSN) are centrally required for skilled voluntary movement, which necessitates that they establish precise subcerebral connectivity with the brainstem and spinal cord. However, molecular controls regulating specificity of this projection targeting remain largely unknown. We previously identified that developing CSN subpopulations exhibit striking axon targeting specificity in the spinal white matter. These CSN subpopulations with segmentally distinct spinal projections are also molecularly distinct; a subset of differentially expressed genes between these distinct CSN subpopulations regulate differential axon projection targeting. Rostrolateral CSN extend axons exclusively to bulbar-cervical segments (CSNBC-lat), while caudomedial CSN (CSNmedial) are more heterogeneous, with distinct, intermingled subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. Here, we report, in male and female mice, that Cerebellin 1 (Cbln1) is expressed specifically by CSN in medial, but not lateral, sensorimotor cortex. Cbln1 shows highly dynamic temporal expression, with Cbln1 levels in CSN highest during the period of peak axon extension toward thoraco-lumbar segments. Using gain-of-function experiments, we identify that Cbln1 is sufficient to direct thoraco-lumbar axon extension by CSN. Misexpression of Cbln1 in CSNBC-lat either by in utero electroporation, or by postmitotic AAV-mediated gene delivery, redirects these axons past their normal bulbar-cervical targets toward thoracic segments. Further, Cbln1 overexpression in postmitotic CSNBC-lat increases the number of CSNmedial axons that extend past cervical segments into the thoracic cord. Collectively, these results identify that Cbln1 functions as a potent molecular control over thoraco-lumbar CSN axon extension, part of an integrated network of controls over segmentally-specific CSN axon projection targeting.SIGNIFICANCE STATEMENT Corticospinal neurons (CSN) exhibit remarkable diversity and precision of axonal projections to targets in the brainstem and distinct spinal segments; the molecular basis for this targeting diversity is largely unknown. CSN subpopulations projecting to distinct targets are also molecularly distinguishable. Distinct subpopulations degenerate in specific motor neuron diseases, further suggesting that intrinsic molecular differences might underlie differential vulnerability to disease. Here, we identify a novel molecular control, Cbln1, expressed by CSN extending axons to thoraco-lumbar spinal segments. Cbln1 is sufficient, but not required, for CSN axon extension toward distal spinal segments, and Cbln1 expression is controlled by recently identified, CSN-intrinsic regulators of axon extension. Our results identify that Cbln1, together with other regulators, coordinates segmentally precise CSN axon targeting.

Three Different Regimens for Vitamin K Birth Prophylaxis in Infants Born Preterm: A Randomized Clinical Trial.

OBJECTIVE: To study the efficacy of 3 different vitamin K birth prophylaxis regimens in infants born premature. STUDY DESIGN: This was an open-label, parallel-group, randomized clinical trial conducted in a tertiary neonatal care unit in India. Infants born very preterm (≤32 weeks) and/or with very low birth weight (≤1500 g) were included. In each arm, 25 babies were enrolled. Babies were randomized to receive 1.0 mg, 0.5 mg, or 0.3 mg intramuscular (IM) vitamin K1 at birth. Protein induced by vitamin K absence - II (PIVKA-II) levels were assessed at birth, and on days 5 and 28, along with the frequency of death, bleeding manifestations, intraventricular hemorrhage, necrotizing enterocolitis, bilirubin levels, and duration of phototherapy. The primary outcome was comparison of PIVKA-II levels on day 5 of life. RESULTS: All the 3 regimens resulted in similar proportion of vitamin K subclinical sufficiency (PIVKA-II < 0.028 AU/mL) infants on day 5 (1 mg - 100%; 0.5 mg - 91.7%; 0.3 mg - 91.7%, P = .347), with no significant difference in median (IQR) PIVKA-II levels (AU/mL): 1 mg 0.006 (0.004, 0.009); 0.5 mg 0.008 (0.004, 0.009); 0.3 mg 0.006 (0.003, 0.009), P = .301. However, on day 28, there was a significant decrease in the proportion of vitamin K-sufficient infants in the 0.3-mg IM group (72.7%) compared with the 1.0-mg (100%) or 0.5-mg (91.3) groups. The 1.0-mg group had significantly greater bilirubin levels and duration of phototherapy. None of the other clinical outcomes were statistically different. CONCLUSIONS: Both 1-mg and 0.5-mg IM vitamin K birth prophylaxis resulted in high sufficiency on follow-up, compared with 0.3 mg. The current recommendation of 0.5-1 mg IM vitamin K birth prophylaxis for infants born preterm, needs to be continued. TRIAL REGISTRATION: CTRI/2022/02/040396.

Cbln1 regulates axon growth and guidance in multiple neural regions.

The accurate construction of neural circuits requires the precise control of axon growth and guidance, which is regulated by multiple growth and guidance cues during early nervous system development. It is generally thought that the growth and guidance cues that control the major steps of axon development have been defined. Here, we describe cerebellin-1 (Cbln1) as a novel cue that controls diverse aspects of axon growth and guidance throughout the central nervous system (CNS) by experiments using mouse and chick embryos. Cbln1 has previously been shown to function in late neural development to influence synapse organization. Here, we find that Cbln1 has an essential role in early neural development. Cbln1 is expressed on the axons and growth cones of developing commissural neurons and functions in an autocrine manner to promote axon growth. Cbln1 is also expressed in intermediate target tissues and functions as an attractive guidance cue. We find that these functions of Cbln1 are mediated by neurexin-2 (Nrxn2), which functions as the Cbln1 receptor for axon growth and guidance. In addition to the developing spinal cord, we further show that Cbln1 functions in diverse parts of the CNS with major roles in cerebellar parallel fiber growth and retinal ganglion cell axon guidance. Despite the prevailing role of Cbln1 as a synaptic organizer, our study discovers a new and unexpected function for Cbln1 as a general axon growth and guidance cue throughout the nervous system.

Distinct neurexin-cerebellin complexes control AMPA- and NMDA-receptor responses in a circuit-dependent manner.

At CA1→subiculum synapses, alternatively spliced neurexin-1 (Nrxn1SS4+) and neurexin-3 (Nrxn3SS4+) enhance NMDA-receptors and suppress AMPA-receptors, respectively, without affecting synapse formation. Nrxn1SS4+ and Nrxn3SS4+ act by binding to secreted cerebellin-2 (Cbln2) that in turn activates postsynaptic GluD1 receptors. Whether neurexin-Cbln2-GluD1 signaling has additional functions besides regulating NMDA- and AMPA-receptors, and whether such signaling performs similar roles at other synapses, however, remains unknown. Here, we demonstrate using constitutive Cbln2 deletions in mice that at CA1→subiculum synapses, Cbln2 performs no additional developmental roles besides regulating AMPA- and NMDA-receptors. Moreover, low-level expression of functionally redundant Cbln1 did not compensate for a possible synapse-formation function of Cbln2 at CA1→subiculum synapses. In exploring the generality of these findings, we examined the prefrontal cortex where Cbln2 was recently implicated in spinogenesis, and the cerebellum where Cbln1 is known to regulate parallel-fiber synapses. In the prefrontal cortex, Nrxn1SS4+-Cbln2 signaling selectively controlled NMDA-receptors without affecting spine or synapse numbers, whereas Nrxn3SS4+-Cbln2 signaling had no apparent role. In the cerebellum, conversely, Nrxn3SS4+-Cbln1 signaling regulated AMPA-receptors, whereas now Nrxn1SS4+-Cbln1 signaling had no manifest effect. Thus, Nrxn1SS4+- and Nrxn3SS4+-Cbln1/2 signaling complexes differentially control NMDA- and AMPA-receptors in different synapses in diverse neural circuits without regulating synapse or spine formation.

Lamin A precursor localizes to the Z-disc of sarcomeres in the heart and is dynamically regulated in muscle cell differentiation.

The lamin A precursor, prelamin A, requires extensive processing to yield mature lamin A and effect its primary function as a structural filament of the nucleoskeleton. When processing is perturbed, nuclear accumulation of prelamin A is toxic and causes laminopathic diseases such as Hutchinson-Gilford progeria syndrome and cardiomyopathy. However, the physiological role of prelamin A is largely unknown and we sought to identify novel insights about this. Using rodent heart tissue, primary cells and the C2C12 model of myofibrillogenesis, we investigated the expression and localization patterns of prelamin A in heart and skeletal muscle cells. We found that endogenous prelamin A was detectable in mouse heart localized to the sarcomere in both adult mouse heart and isolated neonatal rat cardiomyocytes. We investigated the regulation of prelamin A in C2C12 myofibrillogenesis and found it was dynamically regulated and organized into striations upon myofibril formation, colocalizing with the Z-disc protein α-actinin. These data provide evidence that prelamin A is a component of the sarcomere, underpinning a physiological purpose for unprocessed prelamin A. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Diagnostic Role of Tumor Markers for Hepatocellular Carcinoma in Liver Transplantation Candidates: An Analysis Using the Korean Organ Transplantation Registry Database.

BACKGROUND This study analyzed pretransplant alpha-fetoprotein (AFP) and proteins induced by vitamin K absence or antagonist-II (PIVKA-II) in liver transplantation (LT) candidates. MATERIAL AND METHODS A total of 3,273 LT recipients enrolled at the Korean Organ Transplantation Registry were divided according to hepatocellular carcinoma (HCC) status and background liver disease, and AFP and PIVKA-II were compared. RESULTS In all patients, the median AFP and PIVKA-II were 6.3 ng/mL and 29 mAU/mL in the viable-HCC group and 3.3 ng/mL and 35 mAU/mL, respectively, in the no-HCC group (P<0.001 for AFP and p=0.037 for PIVKA-II). In patients with hepatitis B virus infection, they were 6.0 ng/mL and 26 mAU/mL in the HCC group and 3.2 ng/mL and 21 mAU/mL in the no-HCC group, respectively (P<0.001 and P<0.001). In patients with hepatitis C virus infection, they were 10.7 ng/mL and 37 mAU/mL in the HCC group and 2.6 ng/mL and 21 mAU/mL in the no-HCC group, respectively (P<0.001 and P=0.117). In alcoholic liver disease patients, they were 5.2 ng/mL and 61 mAU/mL in the HCC group and 6.4 ng/mL and 75 mAU/mL in the no-HCC group, respectively (P<0.001 and P=0.419). In patients with other diseases, they were 7.1 ng/mL and 32 mAU/mL in the HCC group and 3.3 ng/mL and 28 mAU/mL in the no-HCC group, respectively (P<0.001 and P=0.822). CONCLUSIONS The results of the present study indicate that pretransplant serum AFP and PIVKA-II were highly variably expressed in LT candidates with end-stage liver diseases; therefore, their values should be cautiously interpreted because their role in HCC diagnosis is limited.