Human embryonic stem cells (hESCs) play an important role in regenerative medicine due to their potential to differentiate into various functional cells. However, the conventional adherent culture system poses challenges to mass production of high-quality hESCs. Though scientists have made many attempts to establish a robust and economical hESC suspension culture system, there are existing limitations, including suboptimal passage methods and shear force caused by dynamic stirring. Here, we report on an efficient large-scale culture system, which enables long-term, GMP grade, single-cell inoculation, and serial expansion of hESCs with a yield of about 1.5 × 109 cells per 1.5-L culture, while maintaining good pluripotency. The suspension culture system was enlarged gradually from a 100-mm dish to a 1.8-L culture bag with methylcellulose involvement to avoid sphere fusion. Under the optimal experimental protocol, this 3D system resolves current problems that limit mass production and clinical application of hESCs, and thus can be used in commercial-level hESC production for cell therapy and pharmaceutics screening in the future.

Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer-related death, especially in China. In addition, the prognosis of late stage patients is extremely poor. However, the biological significance of the long non-coding RNA lnc-ATB and its potential role in ESCC remain to be documented. In this study, we investigated the role of lnc-ATB and the underlying mechanism promoting its oncogenic activity in ESCC. Expression of lnc-ATB was higher in ESCC tissues and cell lines than that in normal counterparts. Upregulated lnc-ATB served as an independent prognosis predictor of ESCC patients. Moreover, loss-of-function assays in ESCC cells showed that knockdown of lnc-ATB inhibited cell proliferation and migration both in vitro and in vivo. Mechanistic investigation indicated that lnc-ATB exerted oncogenic activities via regulating Kindlin-2, as the anti-migration role of lnc-ATB silence was attenuated by ectopic expression of Kindlin-2. Further analysis showed that lnc-ATB functions as a molecular sponge for miR-200b and Kindlin-2. Dysregulated miR-200b/Kindlin-2 signaling mediated the oncogenic activity of lnc-ATB in ESCC. Our results suggest that lnc-ATB predicts poor prognosis and may serve as a potential therapeutic target for ESCC patients.

Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans. The first high-resolution structure of fungi rhodopsin and its functional properties reveal the striking similarity of its membrane part to archaeal but not to bacterial rhodopsins. We show that an unusually long N-terminal region stabilizes the protein through direct interaction with its extracellular loop (ECL2). We compare to our knowledge all available structures and sequences of outward light-driven proton pumps and show that eukaryotic and archaeal proton pumps, most likely, share a common ancestor.

The objective of the study is to explore a less invasive laparoscopic pyloromyotomy for treating infantile hypertrophic pyloric stenosis.

The purpose of this study was to investigate the influence of AMP-activated protein kinase (AMPK) activation on protein acetylation and glycolysis in postmortem muscle to better understand the mechanism by which AMPK regulates postmortem glycolysis and meat quality.

Lens opacity seriously affects the visual development of infants. Slit-illumination images play an irreplaceable role in lens opacity detection; however, these images exhibited varied phenotypes with severe heterogeneity and complexity, particularly among pediatric cataracts. Therefore, it is urgently needed to explore an effective computer-aided method to automatically diagnose heterogeneous lens opacity and to provide appropriate treatment recommendations in a timely manner.

Keratitis is the main cause of corneal blindness worldwide. Most vision loss caused by keratitis can be avoidable via early detection and treatment. The diagnosis of keratitis often requires skilled ophthalmologists. However, the world is short of ophthalmologists, especially in resource-limited settings, making the early diagnosis of keratitis challenging. Here, we develop a deep learning system for the automated classification of keratitis, other cornea abnormalities, and normal cornea based on 6,567 slit-lamp images. Our system exhibits remarkable performance in cornea images captured by the different types of digital slit lamp cameras and a smartphone with the super macro mode (all AUCs>0.96). The comparable sensitivity and specificity in keratitis detection are observed between the system and experienced cornea specialists. Our system has the potential to be applied to both digital slit lamp cameras and smartphones to promote the early diagnosis and treatment of keratitis, preventing the corneal blindness caused by keratitis.

Coronavirus disease 2019 (COVID-19) is an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 mainly causes damage to the lung, as well as other organs and systems such as the hearts, the immune system and so on. Although the pathogenesis of COVID-19 has been fully elucidated, there is no specific therapy for the disease at present, and most treatments are limited to supportive care. Stem cell therapy may be a potential treatment for refractory and unmanageable pulmonary illnesses, which has shown some promising results in preclinical studies. In this review, we systematically summarize the pathogenic progression and potential mechanisms underlying stem cell therapy in COVID-19, and registered COVID-19 clinical trials. Of all the stem cell therapies touted for COVID-19 treatment, mesenchymal stem cells (MSCs) or MSC-like derivatives have been the most promising in preclinical studies and clinical trials so far. MSCs have been suggested to ameliorate the cytokine release syndrome (CRS) and protect alveolar epithelial cells by secreting many kinds of factors, demonstrating safety and possible efficacy in COVID-19 patients with acute respiratory distress syndrome (ARDS). However, considering the consistency and uniformity of stem cell quality cannot be quantified nor guaranteed at this point, more work remains to be done in the future.

Coagulase-negative staphylococci (CoNS) for a long time were considered avirulent constituents of the human and warm-blooded animal microbiota. However, at present, S. epidermidis, S. haemolyticus, and S. hominis are recognized as opportunistic pathogens. Although linezolid is not registered for the treatment of CoNS infections, it is widely used off-label, promoting emergence of resistance. Bioinformatic analysis based on maximum-likelihood phylogeny and Bayesian clustering of the CoNS genomes obtained in the current study and downloaded from public databases revealed the existence of international linezolid-resistant lineages, each of which probably had a common predecessor. Linezolid-resistant S. epidermidis sequence-type (ST) 2 from Russia, France, and Germany formed a compact group of closely related genomes with a median pairwise single nucleotide polymorphism (SNP) difference of fewer than 53 SNPs, and a common ancestor of this lineage appeared in 1998 (1986-2006) before introduction of linezolid in practice. Another compact group of linezolid-resistant S. epidermidis was represented by ST22 isolates from France and Russia with a median pairwise SNP difference of 40; a common ancestor of this lineage appeared in 2011 (2008-2013). Linezolid-resistant S. hominis ST2 from Russia, Germany, and Brazil also formed a group with a high-level genome identity with median 25.5 core-SNP differences; the appearance of the common progenitor dates to 2003 (1996-2012). Linezolid-resistant S. hominis isolates from Russia demonstrated associated resistance to teicoplanin. Analysis of a midpoint-rooted phylogenetic tree of the group confirmed the genetic proximity of Russian and German isolates; Brazilian isolates were phylogenetically distant. repUS5-like plasmids harboring cfr were detected in S. hominis and S. haemolyticus.

The performance of deep learning in disease detection from high-quality clinical images is identical to and even greater than that of human doctors. However, in low-quality images, deep learning performs poorly. Whether human doctors also have poor performance in low-quality images is unknown. Here, we compared the performance of deep learning systems with that of cornea specialists in detecting corneal diseases from low-quality slit lamp images. The results showed that the cornea specialists performed better than our previously established deep learning system (PEDLS) trained on only high-quality images. The performance of the system trained on both high- and low-quality images was superior to that of the PEDLS while inferior to that of a senior corneal specialist. This study highlights that cornea specialists perform better in low-quality images than the system trained on high-quality images. Adding low-quality images with sufficient diagnostic certainty to the training set can reduce this performance gap.

Enormous progress has been made in cardiac regeneration using human embryonic stem cell-derived cardiomyocyte (hESC-CM) grafts in pre-clinical trials. However, the rate of cell survival has remained very low due to anoikis after transplantation into the heart as single cells. Numerous solutions have been proposed to improve cell survival, and one of these strategies is to co-transplant biocompatible materials or hydrogels with the hESC-CMs.

Endometriosis is an enigmatic painful disorder whose pain symptoms remain difficult to alleviate in large part because the disorder is defined by extrauteral endometrial growths whose contribution to pain is poorly understood. A rat model (ENDO) involves autotransplanting on abdominal arteries uterine segments that grow into vascularized cysts that become innervated with sensory and sympathetic fibers. ENDO rats exhibit vaginal hyperalgesia. We used behavioral, physiological, and immunohistochemical methods to test the hypothesis that cyst innervation contributes to the development of this hyperalgesia after transplant. Rudimentary sensory and sympathetic innervation appeared in the cysts at two weeks, sprouted further and more densely into the cyst wall by four weeks, and matured by six weeks post-transplant. Sensory fibers became abnormally functionally active between two and three weeks post-transplant, remaining active thereafter. Vaginal hyperalgesia became significant between four and five weeks post-transplant, and stabilized after six to eight weeks. Removing cysts before they acquired functional innervation prevented vaginal hyperalgesia from developing, whereas sham cyst removal did not. Thus, abnormally-active innervation of ectopic growths occurs before hyperalgesia develops, supporting the hypothesis. These findings suggest that painful endometriosis can be classified as a mixed inflammatory/neuropathic pain condition, which opens new avenues for pain relief. The findings also have implications beyond endometriosis by suggesting that functionality of any transplanted tissue can be influenced by the innervation it acquires.

Endometriosis is a disease common in women that is defined by abnormal extrauteral growths of uterine endometrial tissue and associated with severe pain. Partly because how the abnormal growths become associated with pain is poorly understood, the pain is difficult to alleviate without resorting to hormones or surgery, which often produce intolerable side effects or fail to help. Recent studies in a rat model and women showed that sensory and sympathetic nerve fibers sprout branches to innervate the abnormal growths. This situation, together with knowledge that the endocannabinoid system is involved in uterine function and dysfunction and that exogenous cannabinoids were once used to alleviate endometriosis-associated pain, suggests that the endocannabinoid system is involved in both endometriosis and its associated pain. Herein, using a rat model, we found that CB1 cannabinoid receptors are expressed on both the somata and fibers of both the sensory and sympathetic neurons that innervate endometriosis's abnormal growths. We further found that CB1 receptor agonists decrease, whereas CB1 receptor antagonists increase, endometriosis-associated hyperalgesia. Together these findings suggest that the endocannabinoid system contributes to mechanisms underlying both the peripheral innervation of the abnormal growths and the pain associated with endometriosis, thereby providing a novel approach for the development of badly-needed new treatments.

We have successfully generated induced pluripotent stem cells (iPSC) from dermal fibroblasts of a patient with a homozygous p.Leu272Pro mutation in the gene encoding the linear deubiquitinase OTULIN. Biallelic loss of function mutations in this gene are responsible for the OTULIN deficiency termed Otulipenia or OTULIN-related autoinflammatory syndrome (ORAS). The iPSC carrying homozygous L272P OTULIN gene mutations are phenotypically normal and they have capacity to differentiate toward the three germ layers. These iPSC have great potential to study the role of linear ubiquitination in the regulation of immune responses and other cellular pathways.

Autosomal dominant Hyper IgE syndrome (AD-HIES), a rare immune deficiency affecting fewer than one per million people, is caused by heterozygous deleterious mutations in STAT3. STAT3 signaling plays crucial roles in basic cellular functions affecting broad aspects of cellular homeostasis. Accordingly, in addition to immunological deficits, patients experience severe multisystem non-immunological features. Human induced pluripotent stem cells (hiPSC) are well established as in vivo disease models for various human pathologies. We describe the generation of iPSC from three AD-HIES patients. These iPSCs express pluripotency markers, differentiate into three germ layers, have normal karyotype and similar genome identity to parental cells.

We have successfully generated induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells of five patients with Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). These cells carry the genetic NOTCH3 mutation present in their parental cells. These iPSC cells exhibited normal karyotype and phenotype, which were sustained through propagation. Furthermore, these iPSCs displayed the capacity of differentiating toward the three germ layers in vitro.

Immunity-and-matrix-regulatory cells (IMRCs) derived from human embryonic stem cells have unique abilities in modulating immunity and regulating the extracellular matrix, which could be mass-produced with stable biological properties. Despite resemblance to mesenchymal stem cells (MSCs) in terms of self-renew and tri-lineage differentiation, the ability of IMRCs to repair the meniscus and the underlying mechanism remains undetermined. Here, we showed that IMRCs demonstrated stronger immunomodulatory and pro-regenerative potential than umbilical cord MSCs when stimulated by synovial fluid from patients with meniscus injury. Following injection into the knees of rabbits with meniscal injury, IMRCs enhanced endogenous fibrocartilage regeneration. In the dose-escalating phase I clinical trial (NCT03839238) with eighteen patients recruited, we found that intra-articular IMRCs injection in patients was safe over 12 months post-grafting. Furthermore, the effective results of magnetic resonance imaging (MRI) of meniscus repair and knee functional scores suggested that 5 × 107 cells are optimal for meniscus injury treatment. In summary, we present the first report of a phase I clinical trial using IMRCs to treat meniscus injury. Our results demonstrated that intra-articular injection of IMRCs is a safe and effective therapy by providing a permissive niche for cartilage regeneration.

The flow-stimulated intracellular Ca(2+) concentration ([Ca(2+)]i) rise in endothelial cells is an important early event leading to flow-induced blood vessel dilation. Transient receptor potential vanilloid subtype 4 (TRPV4), a Ca(2+)-permeable cation channel, facilitates the flow-stimulated [Ca(2+)]i rise. To determine whether TRPV4 is involved in age-related flow-induced blood vessel dilation impairment, we measured blood vessel diameter and nitric oxide (NO) levels and performed Ca(2+) imaging, immunoblotting, and immunostaining assays in rats. We found that the flow-induced and TRPV4 activator 4α-PDD-induced dilation of mesenteric arteries from aged rats were significantly decreased compared with those from young rats. The flow- or 4α-PDD-induced [Ca(2+)]i rise was also markedly reduced in primary cultured mesenteric artery endothelial cells (MAECs) from aged rats. Immunoblotting and immunostaining results showed an age-related decrease of TRPV4 expression levels in MAECs. Additionally, the 4α-PDD-induced NO production was significantly reduced in aged MAECs. Compared with lentiviral GFP-treated aged rats, lentiviral vector delivery of TRPV4 increased TRPV4 expression level in aged MAECs and restored the flow- and 4α-PDD-induced vessel dilation in aged mesenteric arteries. We concluded that impaired TRPV4-mediated Ca(2+) signaling causes endothelial dysfunction and that TRPV4 is a potential target for clinical treatment of age-related vascular system diseases.

Hepatocellular carcinoma (HCC) is a common malignancy found worldwide and is associated with a high incidence of metastasis and vascular invasion. Elucidating the molecular mechanisms that underlie HCC tumorigenesis and progression is necessary for the development of novel therapeutics. By analyzing the Cancer Genome Atlas Network (TCGA) dataset, we identified Thrombospondin 4 (THBS4) is significantly overexpressed in HCC samples and is correlated with prognosis. Overexpression of THBS4 was also highly correlated with vascular invasion of advanced HCC. While THBS4 is often overexpressed in HCC it has also been shown to inhibit tumor growth by mediating cell-to-cell and cell-to-matrix interactions. Here, we identified that knockdown of THBS4 inhibits migration and invasion of HCC cells and inhibits HCC induced angiogenesis. MiRNAs are crucial regulators of multiple cellular processes, and aberrant expression of miRNAs has been observed to effect cancer development and progression. We further found that miR-142 is an upstream regulator of THBS4 in HCC cells. Moreover, miR-142 was significantly down-regulated in HCC tissue samples and correlated with overexpression of THBS4. Overexpression of miR-142 inhibited invasion and angiogenesis of HCC cells and re-expression of THBS4 overcame these effects of miR-142 expression. Stable over-expression of miR-142 significantly inhibited tumour growth in a xenograft tumour model through inhibiting THBS4 expression and tumor angiogenesis. In conclusion, our findings indicate that loss of miR-142 results in the over-expression of THBS4, which enhances HCC migration and vascular invasion. Thus, targeting THBS4 or miR-142 may provide a promising therapeutic strategy for treatment of advanced HCC.

Chemokine receptor 5 (CCR5) is the primary coreceptor for HIV entry into macrophages. Individuals with a homozygous deletion of 32 bp in the CCR5 gene (CCR5Δ32) are highly resistant to HIV infection (Samson et al., 1996). Allogeneic stem cell transplantation from a healthy donor with the homozygous CCR5Δ32 variant to an HIV positive individual has demonstrated efficient long-term control of HIV. We identified three individuals with this homozygous CCR5Δ32 variant, and successfully generated induced pluripotent stem cell (iPSC) lines from their dermal fibroblasts. The iPSCs lines carrying homozygous CCR5Δ32 variant displayed phenotypically normal and the potential to differentiation toward the three germ layers.