Conditioned medium of human mesenchymal stem cells affects stem cell senescence in osteoporosis.

Systemic transplantation of mesenchymal stem cells (MSCs) and conditioned medium derived from MSCs have been reported to recover bone loss in animal models of osteoporosis; however, the underlying mechanisms remain unclear. We recently reported that extracellular vesicles released from human mesenchymal stem cells (hMSCs) prevent senescence of stem cells in bisphosphonate-related osteonecrosis of the jaw model. In this study, we aimed to compare the effects of conditioned medium (hMSCs-CM) from early and late passage hMSCs on cellular senescence and to verify the benefits of CM from early passage hMSCs in mitigating the progression of osteoporosis through the prevention of cellular senescence. We investigated the distinct endocrine effects of early (P5) and late (P17) passage hMSCs in vitro, as well as the preventive benefits of early passage hMSCs-CM in osteoporosis model triggered by ovariectomy. Our results indicate that long-term cultured hMSCs contributed to the progression of inflammatory transcriptional programs in P5 hMSCs, ultimately impairing their functionality and enhancing senescence-related characteristics. Conversely, early passage hMSCs reversed these alterations. Moreover, early passage hMSCs-CM infused intravenously in a postmenopausal osteoporosis mouse model suppressed bone degeneration and prevented osteoporosis by reducing ovariectomy-induced senescence in bone marrow MSCs and reducing the expression of senescence-associated secretory phenotype-related cytokines. Our findings highlight the high translational value of early passage hMSCs-CM in antiaging intervention and osteoporosis prevention.

Structural basis of hepatitis B virus receptor binding.

Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.

Cryo-EM structures of human zinc transporter ZnT7 reveal the mechanism of Zn2+ uptake into the Golgi apparatus.

Zinc ions (Zn2+) are vital to most cells, with the intracellular concentrations of Zn2+ being tightly regulated by multiple zinc transporters located at the plasma and organelle membranes. We herein present the 2.2-3.1 Å-resolution cryo-EM structures of a Golgi-localized human Zn2+/H+ antiporter ZnT7 (hZnT7) in Zn2+-bound and unbound forms. Cryo-EM analyses show that hZnT7 exists as a dimer via tight interactions in both the cytosolic and transmembrane (TM) domains of two protomers, each of which contains a single Zn2+-binding site in its TM domain. hZnT7 undergoes a TM-helix rearrangement to create a negatively charged cytosolic cavity for Zn2+ entry in the inward-facing conformation and widens the luminal cavity for Zn2+ release in the outward-facing conformation. An exceptionally long cytosolic histidine-rich loop characteristic of hZnT7 binds two Zn2+ ions, seemingly facilitating Zn2+ recruitment to the TM metal transport pathway. These structures permit mechanisms of hZnT7-mediated Zn2+ uptake into the Golgi to be proposed.

Draft Genome Sequence of "Candidatus Liberibacter asiaticus" Strain GZQL4, from Guizhou, China.

Here, we announce the draft genome sequence of "Candidatus Liberibacter asiaticus" strain GZQL4, which was collected from Guizhou, China. The GZQL4 strain has a genome size of 1,234,029 bp, a G+C content of 36.5%, 1,204 predicted open reading frames, and 53 RNA genes.

L-Theanine alleviates heat stress-induced impairment of immune function by regulating the p38 MAPK signalling pathway in mice.

With the current trend of global warming, heat stress-induced impairment could seriously endanger human health. L-Theanine is a non-protein amino acid in tea with various biological activities, including immunoregulatory, anti-anxiety, and anti-oxidation. However, its effect on immune function under heat stress and the underlying mechanism are currently unclear. In this study, male BALB/c mice were used as experimental objects to explore the effect of L-theanine on heat stress-induced changes in immune function and its mechanism. Three doses of L-theanine were used: low (100 mg kg-1 d-1), medium (200 mg kg-1 d-1), and high (400 mg kg-1 d-1). Treatment with L-theanine could attenuate the heat stress-induced reductions in body weight and feed intake in mice, alleviate damage in the liver and jejunum, and inhibit the inflammatory factors IL-6, IL-1ß, and TNF-α. Aspartate aminotransferase and alanine transaminase activity levels and the malondialdehyde content decreased, while the IgA, IgM, and IgG contents increased in response to L-theanine. It is possible that L-theanine affects the P38 signalling pathway and inhibits the increase in p-P65/P65 caused by the overexpression of HSP27 and regulation of PPAR-γ and Foxp3 proteins, thereby alleviating immune dysfunction caused by heat stress.

Genome-wide identification of the trehalose-6-phosphate synthase gene family in sweet orange (Citrus sinensis) and expression analysis in response to phytohormones and abiotic stresses.

Background: Trehalose-6-phosphate synthase (TPS) is an essential enzyme for synthesizing trehalose and is a significant regulator of plant development and stress response. Sweet orange (Citrus sinensis) is an economically important fruit tree crop and a common transgenic material. At present, little information is available about the TPS gene family in sweet orange. Methods: The TPS gene family were identified from sweet orange genome by bioinformatics analysis. Additionally, the expression of CisTPS genes was analyzed under phytohormones and abiotic stresses by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Results: Here, eight TPS genes were identified and were found to be randomly distributed in five sweet orange chromosomes. TPS and trehalose-6-phosphate phosphatase (TPP) domains were observed in all CisTPS proteins. The phylogenetic tree showed that CisTPS genes were divided into two subfamilies, and genes in each subfamily had conserved intron structures and motif compositions. The cis-acting elements of CisTPS genes suggested their roles in phytohormone and stress responses. All CisTPS genes were ubiquitously expressed in roots, leaves, and stems, and six members were highly expressed in roots. Expression profiles showed that CisTPS genes exhibited tissue specificity and were differentially expressed in response to phytohormones and abiotic stresses. This study lays a foundation for revealing the functions of the TPS gene family in trehalose regulation in sweet orange, and provides a valuable reference for this gene family in other plants.

Structure of SARS-CoV-2 membrane protein essential for virus assembly.

The coronavirus membrane protein (M) is the most abundant viral structural protein and plays a central role in virus assembly and morphogenesis. However, the process of M protein-driven virus assembly are largely unknown. Here, we report the cryo-electron microscopy structure of the SARS-CoV-2 M protein in two different conformations. M protein forms a mushroom-shaped dimer, composed of two transmembrane domain-swapped three-helix bundles and two intravirion domains. M protein further assembles into higher-order oligomers. A highly conserved hinge region is key for conformational changes. The M protein dimer is unexpectedly similar to SARS-CoV-2 ORF3a, a viral ion channel. Moreover, the interaction analyses of M protein with nucleocapsid protein (N) and RNA suggest that the M protein mediates the concerted recruitment of these components through the positively charged intravirion domain. Our data shed light on the M protein-driven virus assembly mechanism and provide a structural basis for therapeutic intervention targeting M protein.

Structure of the bile acid transporter and HBV receptor NTCP.

Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually1,2. For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes3. However, the molecular basis for the virus-transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs.

Structural insights into the HBV receptor and bile acid transporter NTCP.

Around 250 million people are infected with hepatitis B virus (HBV) worldwide1, and 15 million may also carry the satellite virus hepatitis D virus (HDV), which confers even greater risk of severe liver disease2. The HBV receptor has been identified as sodium taurocholate co-transporting polypeptide (NTCP), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large protein3. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria4,5, and these models are believed to resemble closely both NTCP and ASBT. Here we have used cryo-electron microscopy to solve the structure of NTCP bound to an antibody, clearly showing that the transporter has no equivalent of the first transmembrane helix found in other SLC10 proteins, and that the N terminus is exposed on the extracellular face. Comparison of our structure with those of related proteins indicates a common mechanism of bile acid transport, but the NTCP structure displays an additional pocket formed by residues that are known to interact with preS1, presenting new opportunities for structure-based drug design.

L-Theanine mediates the p38MAPK signaling pathway to alleviate heat-induced oxidative stress and inflammation in mice.

L-Theanine, an active ingredient in the tea plant (Camellia sinensis) associated with calming, is widely used as a functional ingredient and dietary supplement. In this study, a heat stress mouse model was used to evaluate the anti-heat stress effect of L-theanine and its possible mechanism of action. Mice subjected to heat stress (40 °C) that were administered L-theanine at various doses (100, 200, and 400 mg kg-1 d-1) had reduced oxidative stress and inflammatory factors when L-theanine was administered both long-term and as a preventative treatment. Our L-theanine intervention countered the reduction in growth and feed intake of mice under heat stress and reversed liver and jejunum tissue damage. Moreover, L-theanine countered the increase in inflammatory factors TNF-α, IL-6, and IL-1ß and antioxidant enzymes SOD and CAT; it also counteracted GSH-Px inactivation, the upregulation of AST and ALT enzyme activity, and MDA production. The mechanism of action may involve mediation of the P38 signaling pathway, inhibition of MK2 overexpression, and downregulation of p-P65/P65 caused by the overexpression of downstream HSP27. This would inhibit the heat stress-induced imbalance in oxidative stress and inflammatory responses.

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine.

ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.

Population Diversity of 'Candidatus Liberibacter asiaticus' and Diaphorina citri in Sichuan: A Case Study for Huanglongbing Monitoring and Interception.

Citrus huanglongbing (HLB) is present in 10 provinces in China and is associated with 'Candidatus Liberibacter asiaticus' (CLas), which is transmitted by the Asian citrus psyllid (Diaphorina citri, ACP). To date, HLB and ACP have expanded to Yibin city of Sichuan Province, posing an imminent threat to the citrus belt of the upper and middle reaches of the Yangtze River, an important late-maturing citrus-producing area in China. To understand the epidemiological route of CLas and ACP in newly invaded regions of Sichuan and thereby better establish an HLB interception zone ranging from Leibo to Yibin, we evaluated the molecular variability of 19 CLas draft genomes from citrus or dodder (Cuscuta campestris). They include three type-specific prophage loci, three variable number tandem repeat loci, a miniature inverted-repeat transposable element, and population diversity of 44 ACP mitochondrial genomes. The results indicated that CLas isolates in the newly invaded area (Pingshan) were more diverse than those in the HLB endemic areas (Leibo and Ningnan). Phylogenetic analysis based on mitochondrial genomes demonstrated that ACPs in Leibo, Pingshan, and Xuzhou (rural areas) represent a new mitochondrial group (MG4), distinguished by the three unique single-nucleotide polymorphisms in cox1, nad4, and cytb. However, the ACPs sampled from the urban areas of Cuiping and Xuzhou belonged to the southeastern China group (MG2-1). Altogether, our study revealed multiple sources of ACP and CLas in the HLB interception zone and proposed their transmission route. This study contributes to the formulation of precise HLB prevention and control strategies in the HLB interception zone in Sichuan and could be useful for HLB management efforts in other regions.

Spatio-temporal analysis of EEG features during consciousness recovery in patients with disorders of consciousness.

OBJECTIVE: As consciousness recovery is not only dynamic but also involves interactions between various brain regions, elucidating the mechanism of recovery requires tracking cortical activity in spatio-temporal dimensions. METHODS: We tracked the cortical activities of 40 patients (mean age: 54.38 years; 28 males; 21 patients with minimally conscious states) with disorders of consciousness, and collected a total of 156 electroencephalographic signals. We investigated the longitudinal changes in EEG nonlinear dynamic features (i.e., approximate entropy, sample entropy, and Lempel-Ziv complexity) and relative wavelet energy along with consciousness recovery. RESULTS: Global EEG features showed a non-monotonic trend during consciousness recovery (P < 0.05). When the level of consciousness of patients was transferred to a minimally conscious state from an unresponsive wakefulness syndrome/ vegetative state, an inflection point appeared in the EEG features. The EEG feature change trends between the injured and uninjured areas were dissimilar (P < 0.05). Importantly, the degree of dissimilarity increased non-monotonically across the levels of consciousness (P < 0.05). CONCLUSIONS: EEG recovery was non-monotonic and dissimilar in spatio-temporal dimensions, with an inflection point. SIGNIFICANCE: These findings further clarify the process of consciousness recovery and provide assistance in exploring the mechanism of consciousness recovery.

L-Theanine Regulates the Abundance of Amino Acid Transporters in Mice Duodenum and Jejunum via the mTOR Signaling Pathway.

The intestine is a key organ for the absorption of amino acids. L-theanine (LTA) is a structural analog of glutamine and a characteristic non-protein amino acid found in tea (Camellia sinensis) that regulates lipid and protein metabolism. The present study explored the role of LTA in intestinal amino acid absorption, protein synthesis, and its mechanisms. Overall, our findings suggest that LTA supplementation not only affects serum alkaline phosphatase (AKP), total protein (TP), and urea nitrogen (BUN) levels, but it also upregulates the mRNA and protein expression of amino acid transporters (EAAT3, EAAT1, 4F2hc, y+LAT1, CAT1, ASCT2, and B0AT1), and activates the mTOR signaling pathway. The downstream S6 and S6K1 proteins are regulated, and the expression of amino acid transporters is regulated. These findings suggest that LTA increases intestinal AA absorption, promotes protein metabolism, and increases nitrogen utilization by upregulating AAT expression, activating the mTOR signaling pathway, and phosphorylating the mTOR downstream proteins S6 and S6K1.

Dental pulp stem cell-derived small extracellular vesicle in irradiation-induced senescence.

Small extracellular vesicles (sEV) facilitate signaling molecule transfer among cells. We examined the therapeutic efficacy of human dental pulp stem cell-derived sEV (hDPSC-sEV) against cellular senescence in an irradiated-submandibular gland mouse model. Seven-week-old mice were exposed to 25 Gy radiation and randomly assigned to control, phosphate-buffered saline (PBS), or hDPSC-sEV groups. At 18 days post-irradiation, saliva production was measured; histological and reverse transcription-quantitative PCR analyses of the submandibular glands were performed. The salivary flow rate did not differ significantly between the PBS and hDPSC-sEV groups. AQP5-expressing acinar cell numbers and AQP5 expression levels in the submandibular glands were higher in the hDPSC-sEV group than in the other groups. Furthermore, compared with non-irradiated mice, mice in the 25 Gy + PBS group showed a high senescence-associated-ß-galactosidase-positive cell number and upregulated senescence-related gene (p16INK4a, p19Arf, p21) and senescence-associated secretory phenotypic factor (MMP3, IL-6, PAI-1, NF-κB, and TGF-ß) expression, all of which were downregulated in the hDPSC-sEV group. Superoxide dismutase levels were lower in the PBS group than in the hDPSC-sEV group. In summary, hDPSC-sEV reduced inflammatory cytokine and senescence-related gene expression and reversed oxidative stress in submandibular cells, thereby preventing irradiation-induced cellular senescence. Based on these results, we hope to contribute to the development of innovative treatment methods for salivary gland dysfunction that develops after radiotherapy for head and neck cancer.

The structure of MgtE in the absence of magnesium provides new insights into channel gating.

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

Two Unique Prophages of 'Candidatus Liberibacter asiaticus' Strains from Pakistan.

'Candidatus Liberibacter asiaticus' (CLas) is a pathogen causing Huanglongbing (HLB, yellow shoot disease), which is highly destructive to citrus production. The CLas strains harbor prophages. We identified two unique prophages, designated as P-PA19-1 and P-PA19-2, in CLas strain PA19 from Pakistan using next-generation sequencing analysis. P-PA19-1 prophage has high sequence similarity (identity: 78.23%) at the early-gene region of prophage SC1 (Type 1), but it is significantly divergent in the late-gene region (identity: 62.03%). P-PA19-2 was highly similar to SC2 (Type 2) in the late gene region (identity: 97.96%), and also in the early gene region except for a deletion of a 7,179-bp nucleotide sequence that contains a CRISPR/cas system in SC2. Both P-PA19-1 and P-PA19-2 had circular plasmid forms, and only P-PA19-2 was found integrated in the PA19 chromosome. The two new prophages were only found in Pakistani samples. Identification of prophages enhances our understanding of CLas genomic diversity and also the biology and evolution of CLas prophages.

Genome Sequence Resources of Two 'Candidatus Liberibacter asiaticus' Strains from Pakistan.

'Candidatus Liberibacter asiaticus' (CLas) is an unculturable, phloem-restricted αProteobacteria, associated with citrus Huanglongbing (HLB), which is one of the most destructive diseases in citrus production worldwide. Here, we present the genome sequences of CLas strains PA19 and PA20 from HLB-affected kinnow trees in Multan, Punjab Province, Pakistan. The CLas genomes of PA19 and PA20 comprise 1,224,156 bp and 1,226,225 bp, respectively, with an average GC content of 36.4%. Both harbored the Type 2 prophage. In this study, we report two CLas genomes from Pakistan, which extends the sequence database of CLas and will contribute to CLas biology and HLB management.

Structural insights into tetraspanin CD9 function.

Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short and large extracellular loops, and associate with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2. The reversed cone-like molecular shape of CD9 generates membrane curvature in the crystalline lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The molecular interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the critical involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins.

Outward open conformation of a Major Facilitator Superfamily multidrug/H+ antiporter provides insights into switching mechanism.

Multidrug resistance (MDR) poses a major challenge to medicine. A principle cause of MDR is through active efflux by MDR transporters situated in the bacterial membrane. Here we present the crystal structure of the major facilitator superfamily (MFS) drug/H+ antiporter MdfA from Escherichia coli in an outward open conformation. Comparison with the inward facing (drug binding) state shows that, in addition to the expected change in relative orientations of the N- and C-terminal lobes of the antiporter, the conformation of TM5 is kinked and twisted. In vitro reconstitution experiments demonstrate the importance of selected residues for transport and molecular dynamics simulations are used to gain insights into antiporter switching. With the availability of structures of alternative conformational states, we anticipate that MdfA will serve as a model system for understanding drug efflux in MFS MDR antiporters.