Independent origins of fetal liver haematopoietic stem and progenitor cells.

Self-renewal and differentiation are tightly controlled to maintain haematopoietic stem cell (HSC) homeostasis in the adult bone marrow1,2. During fetal development, expansion of HSCs (self-renewal) and production of differentiated haematopoietic cells (differentiation) are both required to sustain the haematopoietic system for body growth3,4. However, it remains unclear how these two seemingly opposing tasks are accomplished within the short embryonic period. Here we used in vivo genetic tracing in mice to analyse the formation of HSCs and progenitors from intra-arterial haematopoietic clusters, which contain HSC precursors and express the transcription factor hepatic leukaemia factor (HLF). Through kinetic study, we observed the simultaneous formation of HSCs and defined progenitors-previously regarded as descendants of HSCs5-from the HLF+ precursor population, followed by prompt formation of the hierarchical haematopoietic population structure in the fetal liver in an HSC-independent manner. The transcription factor EVI1 is heterogeneously expressed within the precursor population, with EVI1hi cells being predominantly localized to intra-embryonic arteries and preferentially giving rise to HSCs. By genetically manipulating EVI1 expression, we were able to alter HSC and progenitor output from precursors in vivo. Using fate tracking, we also demonstrated that fetal HSCs are slowly used to produce short-term HSCs at late gestation. These data suggest that fetal HSCs minimally contribute to the generation of progenitors and functional blood cells before birth. Stem cell-independent pathways during development thus offer a rational strategy for the rapid and simultaneous growth of tissues and stem cell pools.

Morc1 reestablishes H3K9me3 heterochromatin on piRNA-targeted transposons in gonocytes.

To maintain fertility, male mice re-repress transposable elements (TEs) that were de-silenced in the early gonocytes before their differentiation into spermatogonia. However, the mechanism of TE silencing re-establishment remains unknown. Here, we found that the DNA-binding protein Morc1, in cooperation with the methyltransferase SetDB1, deposits the repressive histone mark H3K9me3 on a large fraction of activated TEs, leading to heterochromatin. Morc1 also triggers DNA methylation, but TEs targeted by Morc1-driven DNA methylation only slightly overlapped with those repressed by Morc1/SetDB1-dependent heterochromatin formation, suggesting that Morc1 silences TEs in two different manners. In contrast, TEs regulated by Morc1 and Miwi2, the nuclear PIWI-family protein, almost overlapped. Miwi2 binds to PIWI-interacting RNAs (piRNAs) that base-pair with TE mRNAs via sequence complementarity, while Morc1 DNA binding is not sequence specific, suggesting that Miwi2 selects its targets, and then, Morc1 acts to repress them with cofactors. A high-ordered mechanism of TE repression in gonocytes has been identified.

Metastasizing aneurysmal dermatofibroma initially diagnosed as angiosarcoma confirmed by CD63::PRKCD fusion gene detection with nanopore sequencing.

Dermatofibroma (DF) is a benign tumor that forms pedunculated lesions ranging in size from a few millimeters to 2 cm, usually affecting the extremities and trunks of young adults. Histopathologically, DF is characterized by the storiform proliferation of monomorphic fibroblast-like spindle cells. In addition to neoplastic cells, secondary elements such as foamy histiocytes, Touton-type giant cells, lymphoplasmacytes, and epidermal hyperplasia are characteristic histological features. Several histological variants, including atypical, cellular, aneurysmal, and lipidized variants, have been reported; cases with variant histologies are sometimes misdiagnosed as sarcomas. We present a case of metastasizing aneurysmal DF that was initially diagnosed as an angiosarcoma on biopsy. A 26-year-old woman was referred to our hospital with a gradually enlarging subcutaneous mass in her lower left leg. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose uptake not only in the tumor but also in the left inguinal region. On biopsy, ERG and CD31-positive atypical spindle cells proliferated in slit-like spaces with extravasation, leading to the diagnosis of angiosarcoma. Histology of the wide-resection specimen was consistent with DF, and lymph node metastasis was also observed. Nanopore DNA sequencing detected CD63::PRKCD fusion and copy number gain, although CD63 was not included in the target region of adaptive sampling. This report highlights the importance of recognizing the unusual clinical, radiological, and pathological features of DF to avoid misdiagnosis, and the potential diagnostic utility of nanopore sequencer.

Loss of Dja2 accompanies pH deviation in lysosomes and lysosome-related organelles.

The Dja2 knockout (Dja2-/- ) mice had respiratory distress, and >60% died within 2 days after birth. The surviving adult Dja2-/- mice were infertile and the lungs of Dja2-/- mice showed several abnormalities, including the processing defect of prosurfactant protein C in the alveolar epithelial type II cells and the accumulation of glycolipids in enlarged alveolar macrophages. The luminal pH of acidic organelles in Dja2-/- cells was shifted to pH 5.37-5.45. This deviated pH was immediately restored to control levels (pH 4.56-4.65) by the addition of a diuretic, ethyl isopropyl amiloride (EIPA). Although the role of DJA2 in maintaining the pH homeostasis of lysosome-related organelles is currently obscure, this rapid and remarkable pH resilience is best explained by an EIPA-sensitive proton efflux machinery that is disorganized and overactivated due to the loss of Dja2.

Author Correction: Meikin is a conserved regulator of meiosis-I-specific kinetochore function.

An Amendment to this Article has been published and is linked from the HTML version of this paper.

Fluorescence in situ hybridization-negative intra-articular myxoid liposarcoma with complex rearrangements involving EWSR1::DDIT3 detected using nanopore sequencing.

Myxoid liposarcoma (MLPS) is a rare sarcoma, typically arising in deep soft tissues during the fourth to fifth decades of life. Histologically, MLPS is composed of uniform oval cells within a background of myxoid stroma and chicken-wire capillaries. Genetically, MLPS is characterized by the FUS/EWSR1::DDIT3 fusion gene, which generally results from balanced interchromosomal translocation and is detectable via DDIT3 break-apart fluorescence in situ hybridization (FISH). Here, we report an unusual intra-articular MLPS case, negative for DDIT3 break-apart FISH but positive for EWSR1::DDIT3. An 18-year-old female was referred to our hospital complaining of an intra-articular mass in the right knee joint. Histologically, the tumor was mainly composed of mature adipocytes, brown fat-like cells, and lipoblasts. Nanopore sequencing detected DNA rearrangements between EWSR1 and DDIT3 and clustered complex rearrangements involving multiple chromosomes, suggesting chromoplexy. Methylation classification using random forest, t-distributed stochastic neighbor embedding, and unsupervised hierarchical clustering correctly classified the tumor as MLPS. The copy number was almost flat. The TERT promoter C-124T was also detected. This report highlights, for the first time, the potential value of a fast and low-cost nanopore sequencer for diagnosing sarcomas.

Metal-free double azide addition to strained alkynes of an octadehydrodibenzo[12]annulene derivative with electron-withdrawing substituents.

Strain-promoted azide-alkyne cycloaddition (SpAAC) is a powerful tool in the field of bioconjugation and materials research. We previously reported a regioselective double addition of organic azides to octadehydrodibenzo[12]annulene derivatives with electron-rich alkyloxy substituents. In order to increase the reaction rate, electron-withdrawing substituents were introduced into octadehydrodibenzo[12]annulene. In this report, the synthesis of new octadehydrodibenzo[12]annulene derivatives, regioselective double addition of organic azides, and an application to crosslinking polymers are described.

Meikin is a conserved regulator of meiosis-I-specific kinetochore function.

The kinetochore is the crucial apparatus regulating chromosome segregation in mitosis and meiosis. Particularly in meiosis I, unlike in mitosis, sister kinetochores are captured by microtubules emanating from the same spindle pole (mono-orientation) and centromeric cohesion mediated by cohesin is protected in the following anaphase. Although meiotic kinetochore factors have been identified only in budding and fission yeasts, these molecules and their functions are thought to have diverged earlier. Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive. Here we have identified in mouse a meiosis-specific kinetochore factor that we termed MEIKIN, which functions in meiosis I but not in meiosis II or mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated mainly by the activity of Polo-like kinase PLK1, which is enriched to kinetochores in a MEIKIN-dependent manner. Our integrative analysis indicates that the long-awaited key regulator of meiotic kinetochore function is Meikin, which is conserved from yeasts to humans.

Recessive mutation in CD2AP causes focal segmental glomerulosclerosis in humans and mice.

Although sequence variants in CD2-associated protein (CD2AP) have been identified in patients with focal segmental glomerulosclerosis (FSGS), definitive proof of causality in human disease is meager. By whole-exome sequencing, we identified a homozygous frame-shift mutation in CD2AP (p.S198fs) in three siblings born of consanguineous parents who developed childhood-onset FSGS and end stage renal disease. When the same frameshift mutation was introduced in mice by gene editing, the mice developed FSGS and kidney failure. These results provide conclusive evidence that homozygous mutation of CD2AP causes FSGS in humans.

Sialyl Tn Unit with TFA-Labile Protection Realizes Efficient Synthesis of Sialyl Glycoprotein.

Amino acids bearing 4-methylbenzyl (MBn) and 4-methoxybenzyl (MPM)-protected sialic acid were synthesized and used for the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of a glycopeptide. The α-sialyl linkage of the MBn-protected unit was partially cleaved under the final deprotection by trifluoroacetic acid (TFA). In addition, the removal of several MBn groups were incomplete. On the other hand, the MPM-protected unit gave the desired glycopeptide without decomposition of the α-sialyl linkage. Using this unit, peptide thioesters of the tandem repeat unit of MUC1 mucin were synthesized by the N-alkylcysteine (NAC) method and used for the one-pot ligation by the thioester method. As a result, the three tandem repeats of MUC1 carrying sialyl Tn antigens were successfully synthesized.

Inflammatory and anti-inflammatory states of adipose tissue in transgenic mice bearing a single TCR.

Obesity is accompanied by chronic, low-grade inflammation in adipose tissue, which is associated with insulin resistance and consequent multiple metabolic diseases. In addition to M1 macrophage infiltration, multiple involvements of adipose tissue T lymphocytes in the progression of inflammation have been highlighted recently. Here, we isolated a specific Vα5/Vß8.2 TCR-bearing T cell that accumulated in obese adipose tissue of mice, and generated transgenic mice expressing this TCR. Under lean conditions with a normal chow diet, CD4+FoxP3+ Treg cells and M2 macrophages increased in adipose tissue with ageing in wild-type mice, but not in transgenic mice. However, both mice exhibited no obvious adipose tissue inflammation such as the formation of crown-like structures (CLSs) of infiltrating macrophages. When fed a high-fat diet, the proportion of adipose tissue Treg cells was markedly small at a similar level in transgenic and wild-type mice. Both types of mice exhibited comparable inflammatory states in adipose tissue, including vast formation of macrophage CLSs, accompanied by insulin resistance. Together, our findings suggest that the absence of an increase in Treg cells and M2 macrophages is not sufficient to initiate inflammatory macrophage infiltration in lean adipose tissue and also provide a new view about the involvement of T cells in promoting obesity-associated inflammation.

Severe ocular phenotypes in Rbp4-deficient mice in the C57BL/6 genetic background.

Retinol-binding protein 4 (RBP4) is a specific carrier for retinol in the blood. In hepatocytes, newly synthesized RBP4 associates with retinol and transthyretin and is secreted into the blood. The ternary transthyretin-RBP4-retinol complex transports retinol in the circulation and delivers it to target tissues. Rbp4-deficient mice in a mixed genetic background (129xC57BL/6J) have decreased sensitivity to light in the b-wave amplitude on electroretinogram. Sensitivity progressively improves and approaches that of wild-type mice at 24 weeks of age. In the present study, we produced Rbp4-deficient mice in the C57BL/6 genetic background. These mice displayed more severe phenotypes. They had decreased a- and b-wave amplitudes on electroretinograms. In accordance with these abnormalities, we found structural changes in these mice, such as loss of the peripheral choroid and photoreceptor layer in the peripheral retinas. In the central retinas, the distance between the inner limiting membrane and the outer plexiform layer was much shorter with fewer ganglion cells and fewer synapses in the inner plexiform layer. Furthermore, ocular developmental defects of retinal depigmentation, optic disc abnormality, and persistent hyaloid artery were also observed. All these abnormalities had not recovered even at 40 weeks of age. Our Rbp4-deficient mice accumulated retinol in the liver but it was undetectable in the serum, indicating an inverse relation between serum and liver retinol levels. Our results suggest that RBP4 is critical for the mobilization of retinol from hepatic storage pools, and that such mobilization is necessary for ocular development and visual function.

Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice.

Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding with-no-lysine kinase 1 (WNK1) and WNK4 kinases are known to be responsible. Recently, Kelch-like 3 (KLHL3) and Cullin3, components of KLHL3-Cullin3 E3 ligase, were newly identified as responsible for PHAII. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and Na-Cl cotransporter (NCC) were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is the target(s) of KLHL3. To investigate the pathogenesis of PHAII caused by KLHL3 mutation, we generated and analyzed KLHL3(R528H/+) knock-in mice. KLHL3(R528H/+) knock-in mice exhibited salt-sensitive hypertension, hyperkalemia and metabolic acidosis. Moreover, the phosphorylation of NCC was increased in the KLHL3(R528H/+) mouse kidney, indicating that the KLHL3(R528H/+) knock-in mouse is an ideal mouse model of PHAII. Interestingly, the protein expression of both WNK1 and WNK4 was significantly increased in the KLHL3(R528H/+) mouse kidney, confirming that increases in these WNK kinases activated the WNK-OSR1/SPAK-NCC phosphorylation cascade in KLHL3(R528H/+) knock-in mice. To examine whether mutant KLHL3 R528H can interact with WNK kinases, we measured the binding of TAMRA-labeled WNK1 and WNK4 peptides to full-length KLHL3 using fluorescence correlation spectroscopy, and found that neither WNK1 nor WNK4 bound to mutant KLHL3 R528H. Thus, we found that increased protein expression levels of WNK1 and WNK4 kinases cause PHAII by KLHL3 R528H mutation due to impaired KLHL3-Cullin3-mediated ubiquitination.

Determining c-Myb protein levels can isolate functional hematopoietic stem cell subtypes.

The transcription factor c-Myb was originally identified as a transforming oncoprotein encoded by two avian leukemia viruses. Subsequently, through the generation of mouse models that affect its expression, c-Myb has been shown to be a key regulator of hematopoiesis, including having critical roles in hematopoietic stem cells (HSCs). The precise function of c-Myb in HSCs although remains unclear. We have generated a novel c-myb allele in mice that allows direct observation of c-Myb protein levels in single cells. Using this reporter line we demonstrate that subtypes of HSCs can be isolated based upon their respective c-Myb protein expression levels. HSCs expressing low levels of c-Myb protein (c-Myb(low) HSC) appear to represent the most immature, dormant HSCs and they are a predominant component of HSCs that retain bromodeoxyuridine labeling. Hematopoietic stress, induced by 5-fluorouracil ablation, revealed that in this circumstance c-Myb-expressing cells become critical for multilineage repopulation. The discrimination of HSC subpopulations based on c-Myb protein levels is not reflected in the levels of c-myb mRNA, there being no more than a 1.3-fold difference comparing c-Myb(low) and c-Myb(high) HSCs. This illustrates how essential it is to include protein studies when aiming to understand the regulatory networks that control stem cell behavior.

The effect of changing toe direction on knee kinematics during drop vertical jump: a possible risk factor for anterior cruciate ligament injury.

PURPOSE: The purpose of this study was to examine the effect of changing toe direction on knee kinetics and kinematics associated with anterior cruciate ligament injury during drop vertical jumps. METHODS: Fourteen females performed drop vertical jumps under three toe conditions (natural, toe-in, and toe-out). The knee kinetics and kinematics during landing were evaluated using a motion analysis system. Results under three toe conditions were compared using a one-way repeated measures analysis of variance and a post hoc Bonferroni test. RESULTS: Toe-in landing was associated with a significantly greater knee abduction angle, tibial internal rotation angle, and knee abduction moment than the natural and toe-out conditions. Toe-out landing was associated with significantly greater tibial internal rotational angular velocity. CONCLUSIONS: Changing toe direction significantly affects knee kinetics and kinematics during landing. It is important to avoid changing toe direction excessively inward or outward during landing to prevent the increases in knee abduction and tibial internal rotation which might increase the risk of ACL injury. LEVEL OF EVIDENCE: Prognosis, Level IV.

Inhibition of PTEN tumor suppressor promotes the generation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) can be generated from patients with specific diseases by the transduction of reprogramming factors and can be useful as a cell source for cell transplantation therapy for various diseases with impaired organs. However, the low efficiency of iPSC derived from somatic cells (0.01-0.1%) is one of the major problems in the field. The phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal, proliferation, and maintenance of embryonic stem cells (ESCs), but the contribution of this pathway or its well-known negative regulator, phosphatase, and tensin homolog deleted on chromosome ten (Pten), to somatic cell reprogramming remains largely unknown. Here, we show that activation of the PI3K pathway by the Pten inhibitor, dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate, improves the efficiency of germline-competent iPSC derivation from mouse somatic cells. This simple method provides a new approach for efficient generation of iPSCs.

The diagnostic utility of cytology specimen in a case of EWSR1::NFATC2 sarcoma.

EWSR1::NFATC2 sarcoma, a rare round cell sarcoma constituting the majority of EWSR1::non-ETS sarcomas, has recently been defined in the latest WHO classification. To date, the cytological findings of EWSR1::NFATC2 sarcoma remain undocumented. We present the case of a 25-year-old man with a history of polyostotic fibrous dysplasia in the right leg, referred to our hospital with left thigh pain. Cytological findings included metachromasia, minimally pleomorphic round cells, and eosinophilic infiltration. There was no precursor fibrous dysplasia and the initial diagnosis was undifferentiated pleomorphic sarcoma. Following histologic review, we successfully performed immunocytochemistry and fluorescence in situ hybridization (FISH) on archival cytology specimens. The tumor cells were positive for NKX2-2, NKX3-1, and PAX7 and showed amplified 5' single signals of EWSR1 gene. Reverse transcriptase-polymerase chain reaction revealed an in-frame fusion of EWSR1 and NFATC2. This report describes the cytological features of EWSR1::NFATC2 sarcoma and highlights the diagnostic utility of archival cytology specimens.

Enhanced inhibitory neurotransmission in the cerebellar cortex of Atp1a3-deficient heterozygous mice.

Dystonia is characterized by excessive involuntary and prolonged simultaneous contractions of both agonist and antagonist muscles. Although the basal ganglia have long been proposed as the primary region, recent studies indicated that the cerebellum also plays a key role in the expression of dystonia. One hereditary form of dystonia, rapid-onset dystonia with parkinsonism (RDP), is caused by loss of function mutations of the gene for the Na pump α3 subunit (ATP1A3). Little information is available on the affected brain regions and mechanism for dystonia by the mutations in RDP. The Na pump is composed of α and ß subunits and maintains ionic gradients of Na(+) and K(+) across the cell membrane. The gradients are utilized for neurotransmitter reuptake and their alteration modulates neural excitability. To provide insight into the molecular aetiology of RDP, we generated and analysed knockout heterozygous mice (Atp1a3(+/-)). Atp1a3(+/-) showed increased symptoms of dystonia that is induced by kainate injection into the cerebellar vermis. Atp1a3 mRNA was highly expressed in Purkinje cells and molecular-layer interneurons, and its product was concentrated at Purkinje cell soma, the site of abundant vesicular γ-aminobutyric acid transporter (VGAT) signal, suggesting the presynaptic localization of the α3 subunit in the inhibitory synapse. Electrophysiological studies showed that the inhibitory neurotransmission at molecular-layer interneuron-Purkinje cell synapses was enhanced in Atp1a3(+/-) cerebellar cortex, and that the enhancement originated via a presynaptic mechanism. Our results shed light on the role of Atp1a3 in the inhibitory synapse, and potential involvement of inhibitory synaptic dysfunction for the pathophysiology of dystonia.

The Rab8 GTPase regulates apical protein localization in intestinal cells.

A number of proteins are known to be involved in apical/basolateral transport of proteins in polarized epithelial cells. The small GTP-binding protein Rab8 was thought to regulate basolateral transport in polarized kidney epithelial cells through the AP1B-complex-mediated pathway. However, the role of Rab8 (Rab8A) in cell polarity in vivo remains unknown. Here we show that Rab8 is responsible for the localization of apical proteins in intestinal epithelial cells. We found that apical peptidases and transporters localized to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes led to a marked reduction in the absorption rate of nutrients in the small intestine, and ultimately to death. Ultrastructurally, a shortening of apical microvilli, an increased number of enlarged lysosomes, and microvillus inclusions in the enterocytes were also observed. One microvillus inclusion disease patient who shows an identical phenotype to Rab8-deficient mice expresses a reduced amount of RAB8 (RAB8A; NM_005370). Our results demonstrate that Rab8 is necessary for the proper localization of apical proteins and the absorption and digestion of various nutrients in the small intestine.

An orally deliverable ornithine-based self-assembling polymer nanomedicine ameliorates hyperammonemia in acetaminophen-induced acute liver injury.

l-Ornithine (Orn) is a core amino acid responsible for ammonia detoxification in the body via the hepatic urea cycle. Clinical studies in Orn therapy have focused on interventions for hyperammonemia-associated diseases, such as hepatic encephalopathy (HE), a life-threatening neurological symptom affecting more than 80% of patients with liver cirrhosis. However, its low molecular weight (LMW) causes Orn to diffuse nonspecifically and be rapidly eliminated from the body after oral administration, resulting in unfavorable therapeutic efficacy. Hence, Orn is constantly supplied by intravenous infusion in many clinical settings; however, this treatment inevitably decreases patient compliance and limits its application in long-term management. To improve the performance of Orn, we designed self-assembling polyOrn-based nanoparticles for oral administration through ring-opening polymerization of Orn-N-carboxy anhydride initiated with amino-ended poly(ethylene glycol), followed by acylation of free amino groups in the main chain of the polyOrn segment. The obtained amphiphilic block copolymers, poly(ethylene glycol)-block-polyOrn(acyl) (PEG-block-POrn(acyl)), enabled the formation of stable nanoparticles (NanoOrn(acyl)) in aqueous media. We employed the isobutyryl (iBu) group for acyl derivatization in this study (NanoOrn(iBu)). In the healthy mice, daily oral administration of NanoOrn(iBu) for one week did not induce any abnormalities. In the mice exhibiting acetaminophen (APAP)-induced acute liver injury, oral pretreatment with NanoOrn(iBu) effectively reduced systemic ammonia and transaminases levels compared to the LMW Orn and untreated groups. The results suggest that the application of NanoOrn(iBu) is of significant clinical value with the feasibility of oral delivery and improvement in APAP-induced hepatic pathogenesis. STATEMENT OF SIGNIFICANCE: Liver injury is often accompanied by hyperammonemia, a life-threatening condition characterized by elevated blood ammonia levels. Current clinical treatments for reducing ammonia typically entail the invasive approach of intravenous infusion, involving the administration of l-ornithine (Orn) or a combination of Orn and L-aspartate. This method is employed due to the poor pharmacokinetics associated with these compounds. In our pursuit of enhancing therapy, we have developed an orally administrable nanomedicine based on Orn-based self-assembling nanoparticle (NanoOrn(iBu)), which provides sustained Orn supply to the injured liver. Oral administration of NanoOrn(iBu) to healthy mice did not cause any toxic effects. In a mouse model of acetaminophen-induced acute liver injury, oral administration of NanoOrn(iBu) surpassed Orn in reducing systemic ammonia levels and liver damage, thereby establishing NanoOrn(iBu) as a safe and effective therapeutic option.