The microRNA408-plantacyanin module balances plant growth and drought resistance by regulating reactive oxygen species homeostasis in guard cells.

The conserved microRNA (miRNA) miR408 enhances photosynthesis and compromises stress tolerance in multiple plants, but the cellular mechanism underlying its function remains largely unclear. Here, we show that in Arabidopsis (Arabidopsis thaliana), the transcript encoding the blue copper protein PLANTACYANIN (PCY) is the primary target for miR408 in vegetative tissues. PCY is preferentially expressed in the guard cells, and PCY is associated with the endomembrane surrounding individual chloroplasts. We found that the MIR408 promoter is suppressed by multiple abscisic acid (ABA)-responsive transcription factors, thus allowing PCY to accumulate under stress conditions. Genetic analysis revealed that PCY elevates reactive oxygen species (ROS) levels in the guard cells, promotes stomatal closure, reduces photosynthetic gas exchange, and enhances drought resistance. Moreover, the miR408-PCY module is sufficient to rescue the growth and drought tolerance phenotypes caused by gain- and loss-of-function of MYB44, an established positive regulator of ABA responses, indicating that the miR408-PCY module relays ABA signaling for regulating ROS homeostasis and drought resistance. These results demonstrate that miR408 regulates stomatal movement to balance growth and drought resistance, providing a mechanistic understanding of why miR408 is selected during land plant evolution and insights into the long-pursued quest of breeding drought-tolerant and high-yielding crops.

ZFYVE19 deficiency: a ciliopathy involving failure of cell division, with cell death.

BACKGROUND AND AIMS: Variants in ZFYVE19 underlie a disorder characterised by progressive portal fibrosis, portal hypertension and eventual liver decompensation. We aim to create an animal model to elucidate the pathogenic mechanism. METHODS: Zfyve19 knockout (Zfyve19-/- ) mice were generated and exposed to different liver toxins. Their livers were characterised at the tissue, cellular and molecular levels. Findings were compared with those in wild-type mice and in ZFYVE19-deficient patients. ZFYVE19 knockout and knockdown retinal pigment epithelial-1 cells and mouse embryonic fibroblasts were generated to study cell division and cell death. RESULTS: The Zfyve19-/- mice were normal overall, particularly with respect to hepatobiliary features. However, when challenged with α-naphthyl isothiocyanate, Zfyve19-/- mice developed changes resembling those in ZFYVE19-deficient patients, including elevated serum liver injury markers, increased numbers of bile duct profiles with abnormal cholangiocyte polarity and biliary fibrosis. Failure of cell division, centriole and cilia abnormalities, and increased cell death were observed in knockdown/knockout cells. Increased cell death and altered mRNA expression of cell death-related signalling pathways was demonstrated in livers from Zfyve19-/- mice and patients. Transforming growth factor-ß (TGF-ß) and Janus kinase-Signal Transducer and Activator of Transcription 3 (JAK-STAT3) signalling pathways were upregulated in vivo, as were chemokines such as C-X-C motif ligands 1, 10 and 12. CONCLUSIONS: Our findings demonstrated that ZFYVE19 deficiency is a ciliopathy with novel histological features. Failure of cell division with ciliary abnormalities and cell death activates macrophages and may thus lead to biliary fibrosis via TGF-ß pathway in the disease.

The PCY-SAG14 phytocyanin module regulated by PIFs and miR408 promotes dark-induced leaf senescence in Arabidopsis.

Leaf senescence is a critical process in plants and has a direct impact on many important agronomic traits. Despite decades of research on senescence-altered mutants via forward genetics and functional assessment of senescence-associated genes (SAGs) via reverse genetics, the senescence signal and the molecular mechanism that perceives and transduces the signal remain elusive. Here, using dark-induced senescence (DIS) of Arabidopsis leaf as the experimental system, we show that exogenous copper induces the senescence syndrome and transcriptomic changes in light-grown plants parallel to those in DIS. By profiling the transcriptomes and tracking the subcellular copper distribution, we found that reciprocal regulation of plastocyanin, the thylakoid lumen mobile electron carrier in the Z scheme of photosynthetic electron transport, and SAG14 and plantacyanin (PCY), a pair of interacting small blue copper proteins located on the endomembrane, is a common thread in different leaf senescence scenarios, including DIS. Genetic and molecular experiments confirmed that the PCY-SAG14 module is necessary and sufficient for promoting DIS. We also found that the PCY-SAG14 module is repressed by a conserved microRNA, miR408, which in turn is repressed by phytochrome interacting factor 3/4/5 (PIF3/4/5), the key trio of transcription factors promoting DIS. Together, these findings indicate that intracellular copper redistribution mediated by PCY-SAG14 has a regulatory role in DIS. Further deciphering the copper homeostasis mechanism and its interaction with other senescence-regulating pathways should provide insights into our understanding of the fundamental question of how plants age.

Tungsten Nitride (W5N6): An Ultraresilient 2D Semimetal.

Two-dimensional transition metal nitrides offer intriguing possibilities for achieving novel electronic and mechanical functionality owing to their distinctive and tunable bonding characteristics compared to other 2D materials. We demonstrate here the enabling effects of strong bonding on the morphology and functionality of 2D tungsten nitrides. The employed bottom-up synthesis experienced a unique substrate stabilization effect beyond van-der-Waals epitaxy that favored W5N6 over lower metal nitrides. Comprehensive structural and electronic characterization reveals that monolayer W5N6 can be synthesized at large scale and shows semimetallic behavior with an intriguing indirect band structure. Moreover, the material exhibits exceptional resilience against mechanical damage and chemical reactions. Leveraging these electronic properties and robustness, we demonstrate the application of W5N6 as atomic-scale dry etch stops that allow the integration of high-performance 2D materials contacts. These findings highlight the potential of 2D transition metal nitrides for realizing advanced electronic devices and functional interfaces.

CD204-positive M2-like tumor-associated macrophages increase migration of gastric cancer cells by upregulating miR-210 to reduce NTN4 expression.

BACKGROUND: Tumor-associated macrophages (TAMs) are the predominant immune cells in the tumor microenvironment and portend poor prognosis. However, the molecular mechanisms underlying the tumor promotion of TAMs have not been fully elucidated. METHODS: Coculture of gastric cancer cells with U937 cells was performed to investigate the impact of TAMs on cancer cell behavior. MicroRNA (miRNA) microarray and bioinformatics were applied to identify the involved miRNAs and the functional target genes. The regulation of the miRNA on its target gene was studied using anti-miRNA and miRNA mimic. RESULTS: Coculture with CD204+ M2-like TAMs increased proliferation, migration, and epithelial-mesenchymal transition of gastric cancer cells. MiR-210 was the most upregulated miRNA in cancer cells identified by miRNA microarray after coculture. In gastric cancer tissues, miR-210 expression was positively correlated with CD204+ M2-like TAM infiltration. Inactivation of miR-210 by antimir attenuated CD204+ M2-like TAMs-induced cancer cell migration. Using pharmacological inhibitors and neutralizing antibodies, CD204+ M2-like TAMs-secreted TNFα was found to upregulate miR-210 through NF-κB/HIF-1α signaling. Bioinformatics analysis showed netrin-4 (NTN4) as a potential target of miR-210 to suppress gastric cancer cell migration. We also found an inverse expression between miR-210 and NTN4 in cancer cells after coculture or in tumor xenografts. Anti-miR-210 increased NTN4 expression, while miR-210 mimics downregulated NTN4 in cancer cells. Reporter luciferase assays showed that MiR-210 mimics suppressed NTN4 3' untranslated region-driven luciferase activity in cancer cells, but this effect was blocked after mutating miR-210 binding site. CONCLUSIONS: CD204+ M2-like TAMs can utilize the TNF-α/NF-κB/HIF-1α/miR-210/NTN4 pathway to facilitate gastric cancer progression.

Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations.

The development of the cerebral cortex involves a series of dynamic events, including cell proliferation and migration, which rely on the motor protein dynein and its regulators NDE1 and NDEL1. While the loss of function in NDE1 leads to microcephaly-related malformations of cortical development (MCDs), NDEL1 variants have not been detected in MCD patients. Here, we identified two patients with pachygyria, with or without subcortical band heterotopia (SBH), carrying the same de novo somatic mosaic NDEL1 variant, p.Arg105Pro (p.R105P). Through single-cell RNA sequencing and spatial transcriptomic analysis, we observed complementary expression of Nde1/NDE1 and Ndel1/NDEL1 in neural progenitors and post-mitotic neurons, respectively. Ndel1 knockdown by in utero electroporation resulted in impaired neuronal migration, a phenotype that could not be rescued by p.R105P. Remarkably, p.R105P expression alone strongly disrupted neuronal migration, increased the length of the leading process, and impaired nucleus-centrosome coupling, suggesting a failure in nucleokinesis. Mechanistically, p.R105P disrupted NDEL1 binding to the dynein regulator LIS1. This study identifies the first lissencephaly-associated NDEL1 variant and sheds light on the distinct roles of NDE1 and NDEL1 in nucleokinesis and MCD pathogenesis.

The PIF1-miR408-PLANTACYANIN repression cascade regulates light-dependent seed germination.

Light-dependent seed germination is a vital process for many seed plants. A decisive event in light-induced germination is degradation of the central repressor PHYTOCHROME INTERACTING FACTOR 1 (PIF1). The balance between gibberellic acid (GA) and abscisic acid (ABA) helps to control germination. However, the cellular mechanisms linking PIF1 turnover to hormonal balancing remain elusive. Here, employing far-red light-induced Arabidopsis thaliana seed germination as the experimental system, we identified PLANTACYANIN (PCY) as an inhibitor of germination. It is a blue copper protein associated with the vacuole that is both highly expressed in mature seeds and rapidly silenced during germination. Molecular analyses showed that PIF1 binds to the miR408 promoter and represses miR408 accumulation. This in turn posttranscriptionally modulates PCY abundance, forming the PIF1-miR408-PCY repression cascade for translating PIF1 turnover to PCY turnover during early germination. Genetic analysis, RNA-sequencing, and hormone quantification revealed that PCY is necessary and sufficient to maintain the PIF1-mediated seed transcriptome and the low-GA-high-ABA state. Furthermore, we found that PCY domain organization and regulation by miR408 are conserved features in seed plants. These results revealed a cellular mechanism whereby PIF1-relayed external light signals are converted through PCY turnover to internal hormonal profiles for controlling seed germination.

Single centre analysis of factors influencing surgical treatment of splenic trauma in children.

OBJECTIVE: This study aims to investigate determinants impacting the surgical management of splenic trauma in paediatric patients by scrutinizing age distribution, etiological factors and concomitant injuries. The analysis seeks to establish a foundation for delineating optimal operative timing. METHODS: A cohort of 262 paediatric cases presenting with splenic trauma at our institution from January 2011 to December 2021 underwent categorization into either the conservative or operative group. RESULTS: Significantly disparate attributes between the two groups included age, time of presentation, blood pressure, haemoglobin levels, blood transfusion requirements, thermal absorption, American Association for the Surgery of Trauma (AAST) classification and associated injuries. Logistic regression analysis revealed age, haemoglobin levels, AAST classification and blood transfusion as autonomous influencers of surgical intervention (OR = 1.024, 95% CI: 1.011-1.037; OR = 1.067, 95% CI: 1.01-1.127; OR = 0.2760, 95% CI: 0.087-0.875; OR = 7.873, 95% CI: 2.442-25.382; OR = 0.016, 95% CI: 0.002-0.153). The AAST type and age demonstrated areas under the receiver operating characteristic (ROC) curve of 0.782 and 0.618, respectively. CONCLUSION: Age, haemoglobin levels, AAST classification and blood transfusion independently influence the decision for surgical intervention in paediatric patients with splenic trauma. Age and AAST classification emerge as viable parameters for assessing and prognosticating the likelihood of surgical intervention in this patient cohort.

Using Sparse Parts in Fused Information to Enhance Performance in Latent Low-Rank Representation-Based Fusion of Visible and Infrared Images.

Latent Low-Rank Representation (LatLRR) has emerged as a prominent approach for fusing visible and infrared images. In this approach, images are decomposed into three fundamental components: the base part, salient part, and sparse part. The aim is to blend the base and salient features to reconstruct images accurately. However, existing methods often focus more on combining the base and salient parts, neglecting the importance of the sparse component, whereas we advocate for the comprehensive inclusion of all three parts generated from LatLRR image decomposition into the image fusion process, a novel proposition introduced in this study. Moreover, the effective integration of Convolutional Neural Network (CNN) technology with LatLRR remains challenging, particularly after the inclusion of sparse parts. This study utilizes fusion strategies involving weighted average, summation, VGG19, and ResNet50 in various combinations to analyze the fusion performance following the introduction of sparse parts. The research findings show a significant enhancement in fusion performance achieved through the inclusion of sparse parts in the fusion process. The suggested fusion strategy involves employing deep learning techniques for fusing both base parts and sparse parts while utilizing a summation strategy for the fusion of salient parts. The findings improve the performance of LatLRR-based methods and offer valuable insights for enhancement, leading to advancements in the field of image fusion.

Nanoscale Gap-Plasmon-Enhanced Superconducting Photon Detectors at Single-Photon Level.

With a growing demand for detecting light at the single-photon level in various fields, researchers are focused on optimizing the performance of superconducting single-photon detectors (SSPDs) by using multiple approaches. However, input light coupling for visible light has remained a challenge in the development of efficient SSPDs. To overcome these limitations, we developed a novel system that integrates NbN superconducting microwire photon detectors (SMPDs) with gap-plasmon resonators to improve the photon detection efficiency to 98% while preserving all detector performance features, such as polarization insensitivity. The plasmonic SMPDs exhibit a hot-belt effect that generates a nonlinear photoresponse in the visible range operated at 9 K (∼0.64Tc), resulting in a 233-fold increase in phonon-electron interaction factor (γ) compared to pristine SMPDs at resonance under CW illumination. These findings open up new opportunities for ultrasensitive single-photon detection in areas like quantum information processing, quantum optics, imaging, and sensing at visible wavelengths.

[Research progress on chemical constituents and pharmacological activities of small molecule compounds in scorpions].

Scorpions, a group of oldest animals with wide distribution in the world, have a long history of medicinal use. Scorpio, the dried body of Buthus martensii, is a rare animal medicine mainly used for the treatment of liver diseases, spasm, and convulsions in children in China. The venom has been considered as the active substance of scorpions. However, little is known about the small molecules in the venom of scorpions. According to the articles published in recent years, scorpions contain amino acids, fatty acids, steroids, and alkaloids, which endow scorpions with antimicrobial, anticoagulant, metabolism-regulating, and antitumor activities. This paper summarizes the small molecule chemical components and pharmacological activities of scorpions, with a view to providing valuable information for the discovery of new active molecules and the clinical use of scorpions.

Miniature Inverted-repeat Transposable Elements Drive Rapid MicroRNA Diversification in Angiosperms.

MicroRNAs (miRNAs) are fast evolving endogenous small RNAs that regulate organism function and behavior in both animals and plants. Although models for de novo miRNA biogenesis have been proposed, the genomic mechanisms driving swift diversification of the miRNA repertoires in plants remain elusive. Here, by comprehensively analyzing 21 phylogenetically representative plant species, ranging from green algae to angiosperms, we systematically identified de novo miRNA events associated with 8,649 miRNA loci. We found that 399 (4.6%), 466 (5.4%), and 1,402 (16.2%) miRNAs were derived from inverted gene duplication events, long terminal repeats of retrotransposons, and miniature inverted-repeat transposable elements (MITEs), respectively. Among the miRNAs of these origins, MITEs, especially those belonging to the Mutator, Tc1/Mariner, and PIF/Harbinger superfamilies, were the predominant genomic source for de novo miRNAs in the 15 examined angiosperms but not in the six non-angiosperms. Our data further illustrated a transposition-transcription process by which MITEs are converted into new miRNAs (termed MITE-miRNAs) whereby properly sized MITEs are transcribed and therefore become potential substrates for the miRNA processing machinery by transposing into introns of active genes. By analyzing the 58,038 putative target genes for the 8,095 miRNAs, we found that the target genes of MITE-miRNAs were preferentially associated with response to environmental stimuli such as temperature, suggesting that MITE-miRNAs are pertinent to plant adaptation. Collectively, these findings demonstrate that molecular conversion of MITEs is a genomic mechanism leading to rapid and continuous changes to the miRNA repertoires in angiosperm.

Exploring Plant Meiosis: Insights from the Kinetochore Perspective.

The central player for chromosome segregation in both mitosis and meiosis is the macromolecular kinetochore structure, which is assembled by >100 structural and regulatory proteins on centromere DNA. Kinetochores play a crucial role in cell division by connecting chromosomal DNA and microtubule polymers. This connection helps in the proper segregation and alignment of chromosomes. Additionally, kinetochores can act as a signaling hub, regulating the start of anaphase through the spindle assembly checkpoint, and controlling the movement of chromosomes during anaphase. However, the role of various kinetochore proteins in plant meiosis has only been recently elucidated, and these proteins differ in their functionality from those found in animals. In this review, our current knowledge of the functioning of plant kinetochore proteins in meiosis will be summarized. In addition, the functional similarities and differences of core kinetochore proteins in meiosis between plants and other species are discussed, and the potential applications of manipulating certain kinetochore genes in meiosis for breeding purposes are explored.

Ultracompact Vernier-effect-improved sensor by a single microfiber-knot resonator.

Fiber-optic sensors are an indispensable element of modern sensing technologies by virtue of their low cost, excellent electromagnetic immunity, and remote sensing capability. Optical Vernier effect is widely used to enhance sensitivity of fiber-optic sensors but requires bulky and complex cascaded interferometers. Here we propose and experimentally demonstrate an ultracompact (∼2 mm by ∼2 mm) Vernier-effect-improved sensor by only using a single microfiber-knot resonator. With the Vernier effect achieved by controlling the optical beating with the spectral ripple of a super light emitting diode (SLED), we show ∼20x sensitivity enhancement for quantitative temperature monitoring. Our sensor creates a new practical method to realize Vernier effect in fiber-optic sensors and beyond.

Improving the performance of reference-frame-independent quantum key distribution with advantage distillation technology.

The reference-frame-independent quantum key distribution (RFI-QKD) has the advantage of tolerating reference frames that slowly vary. It can generate secure keys between two remote users with slowly drifted and unknown reference frames. However, the drift of reference frames may inevitably compromise the performance of QKD systems. In the paper, we employ the advantage distillation technology (ADT) to the RFI-QKD and the RFI measurement-device-independent QKD (RFI MDI-QKD), and we then analyze the effect of ADT on the performance of decoy-state RFI-QKD and RFI MDI-QKD in both asymptotic and nonasymptotic cases. The simulation results show that ADT can significantly improve the maximum transmission distance and the maximum tolerable background error rate. Furthermore, the performance of RFI-QKD and RFI MDI-QKD in terms of the secret key rate and maximum transmission distance are still greatly improved when statistical fluctuations are taken into account. Our work combines the merits of the ADT and RFI-QKD protocols, which further enhances the robustness and practicability of QKD systems.

Regulatory roles of microRNA163 in responses to stresses in Arabidopsis.

MicroRNAs (miRNAs) are small regulatory RNAs that participate in various biological processes by silencing target genes. In Arabidopsis, microRNA163 (miR163) was found to be involved in seed germination, root development, and biotic resistance. However, the regulatory roles of miR163 remain unclear. In the current study, the mir163 mutant was investigated to comprehensively understand and characterize its functions in Arabidopsis. RNA-sequencing and Gene Ontology enrichment analyses revealed that miR163 might be involved in "response to stimulus" and "metabolic process". Interestingly, "response to stress", including heat, cold, and oxidative stress, was enriched under the subcategory of "response to stimulus". We observed that miR163 and PXMT were repressed and induced under heat stress, respectively. Furthermore, the study detected significant differences in seed germination rate, hypocotyl length, and survival rate, indicating a variation in the thermotolerance between WT and mir163 mutant. The results revealed that the mir163 mutant had a lesser degree of germination inhibition by heat treatment than WT. In addition, the mir163 mutant showed a better survival rate and longer hypocotyl length under heat treatment than the WT. The metabolomes of WT and mir163 mutant were further analyzed. The contents of benzene derivatives and flavonoids were affected by miR163, which could enhance plants' defense abilities. In conclusion, miR163/targets regulated the expression of stress-responsive genes and the accumulation of defense-related metabolites to alter stress tolerance.

Programmable DNA hydrogel provides suitable microenvironment for enhancing autophagy-based therapies in intervertebral disc degeneration treatment.

The pathogenesis of intervertebral disc degeneration (IVDD) is attributed to metabolic dysregulation within the extracellular matrix and heightened apoptosis of nucleus pulposus cells (NPC). Therefore, a potential therapeutic strategy for managing IVDD involves the reestablishment of metabolic equilibrium within the extracellular matrix and the suppression of excessive myeloid cell apoptosis. The microRNA, miR-5590, displays marked differential expression in degenerative nucleus pulposus (NP) tissues and exerts a direct influence on the regulation of DDX5 expression. This, in turn, modulates mammalian target of rapamycin (mTOR) phosphorylation, thereby impacting autophagy and apoptosis. However, ensuring the smooth delivery of miRNA to a specific injury site poses a significant challenge. To address this issue, a multifunctional DNA hydrogel was developed and subsequently loaded with miR-5590 via spherical nucleic acids (SNAs) for the treatment of IVDD. The hydrogel, which exhibits versatility, has the potential to be administered through injection at the site of injury, resulting in a consistent and prolonged release of miR-5590. This leads to the creation of a genetic microenvironment within the NP, which triggers the onset of autophagy in NPCs and subsequently suppresses apoptosis. As a result, this process regulates the metabolic equilibrium within the extracellular matrix, thereby impeding the in vitro and in vivo progression of IVDD. The amalgamation of miRNAs and biomaterials offers a promising therapeutic strategy for the management of IVDD in clinical settings.

Face mask-derived Ni, N-doped graphene sheets for electrocatalytic CO2-to-CO reduction.

The COVID-19 pandemic that is still prevalent around the globe each day consumes massive disposable face masks and consequently lays a heavy burden on waste management. Meanwhile, the incineration of these medical wastes further escalates the already overwhelming carbon emission that leads to global warming and climate change. To offer a potential solution addressing medical waste and CO2 emission challenges, we herein develop a synthetic protocol to upgrade face masks into Ni, N-doped graphene (Ni-N-C) sheet catalysts for selectively reducing CO2 into CO electrochemically. The high specific surface area and the uniform dispersion of Ni active sites of the catalyst derived from a regular disposable face mask enable a near-unity CO Faradaic efficiency (FE) at the current density of 200 mA cm-2. This study offers outside-of-the-box thinking to address environmental issues by turning medical wastes into CO2 reduction catalysts. Supplementary Information: The online version contains supplementary material available at 10.1007/s10008-023-05444-7.

A Comprehensive Genetic Study of Microtubule-Associated Gene Clusters for Male Infertility in a Taiwanese Cohort.

Advanced reproductive technologies are utilized to identify the genetic mutations that lead to spermatogenic impairment, and allow informed genetic counseling to patients to prevent the transmission of genetic defects to offspring. The purpose of this study was to identify potential single nucleotide polymorphisms (SNPs) associated with male infertility. Genetic variants that may cause infertility are identified by combining the targeted next-generation sequencing (NGS) panel and whole exome sequencing (WES). The validation step of Sanger sequencing adds confidence to the identified variants. Our analysis revealed five distinct affected genes covering seven SNPs based on the targeted NGS panel and WES data: SPATA16 (rs16846616, 1515442, 1515441), CFTR (rs213950), KIF6 (rs2273063), STPG2 (r2903150), and DRC7 (rs3809611). Infertile men have a higher mutation rate than fertile men, especially those with azoospermia. These findings strongly support the hypothesis that the dysfunction of microtubule-related and spermatogenesis-specific genes contributes to idiopathic male infertility. The SPATA16, CFTR, KIF6, STPG2, and DRC7 mutations are associated with male infertility, specifically azoospermia, and a further examination of this genetic function is required.

Association of novel TMEM67 variants with mild phenotypes of high gamma-glutamyl transpeptidase cholestasis and congenital hepatic fibrosis.

TMEM67 (mecklin or MKS3) locates in the transition zone of cilia. Dysfunction of TMEM67 disrupts cilia-related signaling and leads to developmental defects of multiple organs in humans. Typical autosomal recessive TMEM67 defects cause partial overlapping phenotypes, including abnormalities in the brain, eyes, liver, kidneys, bones, and so forth. However, emerging reports of isolated nephronophthisis suggest the possibility of a broader phenotype spectrum. In this study, we analyzed the genetic data of cholestasis patients with no obvious extrahepatic involvement but with an unexplained high level of gamma-glutamyl transpeptidase (GGT). We identified five Han Chinese patients from three unrelated families with biallelic nonnull low-frequency TMEM67 variants. All variants were predicted pathogenic in silico, of which p. Arg820Ile and p. Leu144del were previously unreported. In vitro studies revealed that the protein levels of the TMEM67 variants were significantly decreased; however, their interaction with MKS1 remained unaffected. All the patients, aged 7-39 years old, had silently progressive cholestasis with elevated GGT but had normal bilirubin levels. Histological studies of liver biopsy of patients 1, 3, and 5 showed the presence of congenital hepatic fibrosis. We conclude that variants in TMEM67 are associated with a mild phenotype of unexplained, persistent, anicteric, and high GGT cholestasis without typical symptoms of TMEM67 defects; this possibility should be considered by physicians in gastroenterology and hepatology.