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The modification of an endogenous gene into a designed sequence by homologous recombination, termed gene targeting (GT), has broad implications for basic and applied research. Rice (Oryza sativa), with a sequenced genome of 389 Mb, is one of the most important crops and a model plant for cereals, and the single-copy gene Waxy on chromosome 6 has been modified with a frequency of 1% per surviving callus by GT using a strong positive-negative selection. Because the strategy is independent of gene-specific selection or screening, it is in principle applicable to any gene. However, a gene in the multigene family or a gene carrying repetitive sequences may preclude efficient homologous recombination-promoted GT due to the occurrence of ectopic recombination. Here, we describe an improved GT procedure whereby we obtained nine independent transformed calli having the alcohol dehydrogenase2 (Adh2) gene modified with a frequency of approximately 2% per surviving callus and subsequently isolated eight fertile transgenic plants without the concomitant occurrence of undesirable ectopic events, even though the rice genome carries four Adh genes, including a newly characterized Adh3 gene, and a copy of highly repetitive retroelements is present adjacent to the Adh2 gene. The results indicate that GT using a strong positive-negative selection can be widely applicable to functional genomics in rice and presumably in other higher plants.

Hepatocytes derived from human embryonic stem cells (hESCs) are an attractive cell source for regenerative medicine. We previously reported the differentiation of hESCs into alpha-fetoprotein (AFP)-producing endodermal cells by using extracellular matrix and growth factors. We also reported the establishment of the MLSgt20 cell line, which was derived from mesenchymal cells residing in murine fetal livers and accelerated the hepatic maturation of both murine hepatic progenitor cells and murine ESCs. In this study, hESC-derived AFP-producing cells were isolated by using a flow cytometer and co-cultured with MLSgt20 cells. The co-cultured hESC-derived AFP-producing cells had the immunocytological characteristics of hepatocytes, expressed mature hepatocyte markers (as indicated by reverse transcription and the polymerase chain reaction), and displayed higher hepatocyte functions including ammonia removal, cytochrome P450 3A4/7 activity, and the ability to produce and store glycogen. However, the MLSgt20 cells did not directly cause undifferentiated hESCs to mature into hepatocyte-like cells. The co-culture method was thus successfully shown to induce the differentiation of hESC-derived endodermal cells into functional hepatocyte-like cells.

The modification of an endogenous gene into a designed sequence by homologous recombination, termed gene targeting (GT), has broad implications for basic and applied research. Rice (Oryza sativa), with a sequenced genome of 389 Mb, is one of the most important crops and a model plant for cereals, and the single-copy gene Waxy on chromosome 6 has been modified with a frequency of 1% per surviving callus by GT using a strong positive-negative selection. Because the strategy is independent of gene-specific selection or screening, it is in principle applicable to any gene. However, a gene in the multigene family or a gene carrying repetitive sequences may preclude efficient homologous recombination-promoted GT due to the occurrence of ectopic recombination. Here, we describe an improved GT procedure whereby we obtained nine independent transformed calli having the alcohol dehydrogenase2 (Adh2) gene modified with a frequency of approximately 2% per surviving callus and subsequently isolated eight fertile transgenic plants without the concomitant occurrence of undesirable ectopic events, even though the rice genome carries four Adh genes, including a newly characterized Adh3 gene, and a copy of highly repetitive retroelements is present adjacent to the Adh2 gene. The results indicate that GT using a strong positive-negative selection can be widely applicable to functional genomics in rice and presumably in other higher plants.

Cranial nerve involvement is a finding often observed in patients infected with severe acute respiratory syndrome coronavirus 2 during the pandemic outbreak of coronavirus disease 2019 (COVID-19). To our knowledge, this is the first report of oropharyngeal dysphagia associated with COVID-19. A 70-year-old male developed dysphagia and consequent aspiration pneumonia during recovery from severe COVID-19. He had altered sense of taste and absent gag reflex. Videoendoscopy, videofluorography, and high-resolution manometry revealed impaired pharyngolaryngeal sensation, silent aspiration, and mesopharyngeal contractile dysfunction. These findings suggested that glossopharyngeal and vagal neuropathy might have elicited dysphagia following COVID-19. The current case emphasizes the importance of presuming neurologic involvement and concurrent dysphagia, and that subsequent aspiration pneumonia might be overlooked in severe respiratory infection during COVID-19.

Non-polyadenylated mRNAs of replication-dependent histones (RDHs) are synthesized by RNA polymerase II (Pol II) at histone locus bodies (HLBs). HLBs frequently associate with Cajal bodies (CBs), in which 3′-end processing factors for RDH genes are enriched; however, this association’s role in transcription termination of RDH genes remains unclear. Here, we show that Pol II pauses immediately upstream of transcript end sites of RDH genes and Mediator plays a role in this Pol II pausing through CBs’ association with HLBs. Disruption of the Mediator docking site for Little elongation complex (LEC)–Cap binding complex (CBC)–Negative elongation factor (NELF), components of CBs, interferes with CBs’ association with HLBs and 3′ Pol II pausing, resulting in increased aberrant unprocessed RDH gene transcripts. Our findings suggest Mediator’s involvement in CBs’ association with HLBs to facilitate 3′ Pol II pausing and subsequent 3′-end processing of RDH genes by supplying 3′-end processing factors. Transcription termination is generally accompanied by the 3′-end processing of transcripts. Here the authors demonstrate a role for Mediator in transcript end site proximal pausing of replication-dependent histone genes.

In response to signals from afferent TRPV1 + C-fibers, STAT3-dependent upregulation of LCN2 in reactive astrocytes of the spinal dorsal horn amplifies itch signaling in multiple rodent models of atopic and contact dermatitis. Chronic itch is an intractable symptom of inflammatory skin diseases, such as atopic and contact dermatitis 1 , 2 , 3 . Recent studies have revealed neuronal pathways selective for itch 4 , 5 , 6 , 7 , 8 , but the mechanisms by which itch turns into a pathological chronic state are poorly understood. Using mouse models of atopic and contact dermatitis, we demonstrate a long-term reactive state of astrocytes in the dorsal horn of the spinal segments that corresponds to lesioned, itchy skin. We found that reactive astrogliosis depended on the activation of signal transducer and activator of transcription 3 (STAT3). Conditional disruption of astrocytic STAT3 suppressed chronic itch, and pharmacological inhibition of spinal STAT3 ameliorated the fully developed chronic itch. Mice with atopic dermatitis exhibited an increase in scratching elicited by intrathecal administration of the itch-inducer gastrin-releasing peptide (GRP) 4 , and this enhancement was normalized by suppressing STAT3-mediated reactive astrogliosis. Moreover, we identified lipocalin-2 (LCN2) as an astrocytic STAT3-dependent upregulated factor that was crucial for chronic itch, and we demonstrated that intrathecal administration of LCN2 to normal mice increased spinal GRP-evoked scratching. Our findings indicate that STAT3-dependent reactive astrocytes act as critical amplifiers of itching through a mechanism involving the enhancement of spinal itch signals by LCN2, thereby providing a previously unrecognized target for treating chronic itch.

Tongue-hold swallow (THS) is a swallow exercise in which an individual swallows saliva while holding the anterior portion of the tongue between the front teeth. The effect of THS on pharyngeal contractile vigor is still unclear. The purpose of this study was to quantify THS using high-resolution manometry with a contractile integral analysis. Twenty-two healthy participants performed three different saliva swallow tasks: normal swallow, weak THS (in which the tongue was protruded 1 cm outside the upper incisors), and strong THS (in which the tongue was protruded 2 cm outside the upper incisors). The participants repeated each task twice randomly. Pharyngeal and upper esophageal sphincter metrics, including the pharyngeal contractile integral, were analyzed. Both weak and strong THS enhanced the velopharyngeal contractile integral and peak pressure compared with normal swallow (P < 0.01). THS also prolonged mesopharyngeal contraction (P < 0.01). Holding the tongue anteriorly during swallow requires significant biomechanical changes to pharyngeal contractile properties at the superior and middle pharyngeal constrictor levels; thus, it may serve as a resistance exercise for the muscles that are involved in bolus propulsion.

P2X4 receptors (P2X4R) are a family of ATP-gated non-selective cation channels. We previously demonstrated that activation of P2X4R in spinal microglia is crucial for neuropathic pain, a highly debilitating chronic pain condition, suggesting that P2X4R is a potential therapeutic target for treating neuropathic pain. Thus, the identification of a compound that has a potent inhibitory effect on P2X4R is an important clinical challenge. In the present study, we screened a chemical library of clinically approved drugs and show for the first time that duloxetine, a serotonin and noradrenaline reuptake inhibitor, has an inhibitory effect on rodent and human P2X4R. In primary cultured microglial cells, duloxetine also inhibited P2X4R-, but not P2X7R-, mediated responses. Moreover, intrathecal administration of duloxetine in a model of neuropathic pain produced a reversal of nerve injury-induced mechanical allodynia, a cardinal symptom of neuropathic pain. In rats that were pretreated with a serotonin-depleting agent and a noradrenaline neurotoxin, the antiallodynic effect of duloxetine was reduced, but still remained. Based on these results, we suggest that, in addition to duloxetine's primary inhibitory action on serotonin and noradrenaline transporters, an inhibitory effect on P2X4R may be involved at least in part in an antiallodynic effect of intrathecal duloxetine in a model of neuropathic pain.

To bioengineer ectodermal organs such as teeth and whisker follicles, we developed a three-dimensional organ-germ culture method. The bioengineered tooth germ generated a structurally correct tooth, after both in vitro organ culture as well as transplantation under a tooth cavity in vivo , showing penetration of blood vessels and nerve fibers. Our method provides a substantial advance in the development of bioengineered organ replacement strategies and regenerative therapies. Please visit methagora to view and post comments on this article

Accumulating evidence indicates that peripheral nerve injury (PNI) activates spinal microglia that are necessary for neuropathic pain. Recent studies using bone marrow (BM) chimeric mice have reported that after PNI, circulating BM-derived cells infiltrate into the spinal cord and differentiate into microglia-like cells. This raises the possibility that the population of spinal microglia after PNI may be heterogeneous. However, the infiltration of BM cells in the spinal cord remains controversial because of experimental adverse effects of strong irradiation used for generating BM chimeric mice. In this study, we evaluated the PNI-induced spinal infiltration of BM-derived cells not only by irradiation-induced myeloablation with various conditioning regimens, but also by parabiosis and mice with genetically labelled microglia, models without irradiation and BM transplantation. Results obtained from these independent approaches provide compelling evidence indicating little contribution of circulating BM-derived cells to the population of spinal microglia after PNI.