Hemoglobin is associated with BMDs and risk of the 10-year probability of fractures in patients with type 2 diabetes mellitus.

Purpose: This study aimed to investigate the associations between hemoglobin (HGB) levels and bone mineral density (BMD) and fracture risk in type 2 diabetes mellitus(T2DM) population of different ages. Method: This cross-sectional study included 641 patients with T2DM (57.9% males). BMD of the femoral neck (FN), total hip (TH), and lumbar spine (LS) were measured using dual-energy X-ray absorptiometry. The 10-year probability of fracture was assessed using a fracture risk assessment tool (FRAX). HGB and other biochemical indices were measured in a certified laboratory at our hospital. Statistical analysis was performed using SPSS 26.0 and R language (R version 4.1.0). Generalized additive models (GAMs) were used to identify the associations between HGB and BMD and fracture risk. Results: Patients with osteoporosis have lower HGB levels than the non-osteoporotic population and lower FN BMD in patients with anemia than in the non-anemic population. In patients with T2DM, there was sex- and age-related variability in the correlation between HGB levels and BMDs and fracture risk. In older men, HGB level was an independent determinant of BMD and was positively correlated with FN and TH BMD. In non-older women, HGB level was an independent determinant of BMD and fracture risk, positively associated with BMDs and negatively associated with 10-year probability of fracture risk. GAMs revealed a positive linear association between HGB level and BMDs in non-older female patients but not in older male patients. Conclusion: Our study provides a new perspective on the association of HGB level and BMDs with fracture risk. Relatively high HGB levels are a protective factor for bone quality in patients with T2DM. However, the bone-protective effect of HGB is influenced by age and sex and persists only in older men and non-older women with T2DM.

Structural basis for the ubiquitination of G protein ßγ subunits by KCTD5/Cullin3 E3 ligase.

G protein-coupled receptor (GPCR) signaling is precisely controlled to avoid overstimulation that results in detrimental consequences. Gßγ signaling is negatively regulated by a Cullin3 (Cul3)-dependent E3 ligase, KCTD5, which triggers ubiquitination and degradation of free Gßγ. Here, we report the cryo-electron microscopy structures of the KCTD5-Gßγ fusion complex and the KCTD7-Cul3 complex. KCTD5 in pentameric form engages symmetrically with five copies of Gßγ through its C-terminal domain. The unique pentameric assembly of the KCTD5/Cul3 E3 ligase places the ubiquitin-conjugating enzyme (E2) and the modification sites of Gßγ in close proximity and allows simultaneous transfer of ubiquitin from E2 to five Gßγ subunits. Moreover, we show that ubiquitination of Gßγ by KCTD5 is important for fine-tuning cyclic adenosine 3´,5´-monophosphate signaling of GPCRs. Our studies provide unprecedented insights into mechanisms of substrate recognition by unusual pentameric E3 ligases and highlight the KCTD family as emerging regulators of GPCR signaling.

Somatic genetics analysis of sleep in adult mice.

Classical forward and reverse mouse genetics require germline mutations and, thus, are unwieldy to study sleep functions of essential genes or redundant pathways. It is also time-consuming to conduct electroencephalogram/electromyogram-based mouse sleep screening owing to labor-intensive surgeries and genetic crosses. Here, we describe a highly accurate SleepV (video) system and adeno-associated virus (AAV)-based adult brain chimeric (ABC)-expression/knockout (KO) platform for somatic genetics analysis of sleep in adult male or female mice. A pilot ABC screen identifies CREB and CRTC1, of which constitutive or inducible expression significantly reduces quantity and/or quality of non-rapid eye movement sleep. Whereas ABC-KO of exon 13 of Sik3 by AAV-Cre injection in Sik3-E13flox/flox adult mice phenocopies Sleepy (Sik3Slp/+) mice, ABC-CRISPR of Slp/Sik3 reverses hypersomnia of Sleepy mice, indicating a direct role of SLP/SIK3 kinase in sleep regulation. Multiplex ABC-CRISPR of both orexin/hypocretin receptors causes narcolepsy episodes, enabling one-step analysis of redundant genes in adult mice. Therefore, this somatic genetics approach should facilitate high-throughput analysis of sleep regulatory genes, especially for essential or redundant genes, in adult mice by skipping mouse development and minimizing genetic crosses.SIGNIFICANCE STATEMENTThe molecular mechanisms of mammalian sleep regulation remain unclear. Classical germline mouse genetics are unwieldy to study sleep functions of essential genes or redundant pathways. The EEG/EMG-based mouse sleep screening is time-consuming owing to labor-intensive surgeries and lengthy genetic crosses. To overcome these "bottlenecks", we developed a highly accurate video-based sleep analysis system and adeno-associated virus-mediated ABC-expression/knockout platform for somatic genetics analysis of sleep in adult mice. These methodologies facilitate rapid identification of sleep regulatory genes, but also efficient mechanistic studies of the molecular pathways of sleep regulation in mice.

Structural insights into galanin receptor signaling.

Galanin is a biologically active neuropeptide, and functions through three distinct G protein­coupled receptors (GPCRs), namely GALR1, GALR2, and GALR3. GALR signaling plays important roles in regulating various physiological processes such as energy metabolism, neuropathic pain, epileptic activity, and sleep homeostasis. GALR1 and GALR3 signal through the Gi/o pathway, whereas GALR2 signals mainly through the Gq/11 pathway. However, the molecular basis for galanin recognition and G protein selectivity of GALRs remains poorly understood. Here, we report the cryoelectron microscopy structures of the GALR1-Go and the GALR2-Gq complexes bound to the endogenous ligand galanin or spexin. The galanin peptide mainly adopts an alpha helical structure, which binds at the extracellular vestibule of the receptors, nearly parallel to the membrane plane without penetrating deeply into the receptor core. Structural analysis combined with functional studies reveals important structural determinants for the G protein selectivity of GALRs as well as other class A GPCRs. In addition, we show that the zinc ion is a negative allosteric regulator of GALR1 but not GALR2. Our studies provide insight into the mechanisms of G protein selectivity of GPCRs and highlight a potential function of the neuromodulator zinc ion as a modulator of GPCR signaling in the central nervous system.

Three kinds of corneal host cells contribute differently to corneal neovascularization.

BACKGROUND: Corneal neovascularization (angiogenesis and lymphangiogenesis) compromises corneal transparency and transplant survival, however, the molecular mechanisms of corneal host epithelial and stromal cells in neovascularization have not yet been fully elucidated. Furthermore, the contribution and mechanism of corneal host endothelial cells involved in neovascularization are largely unexplored. METHODS: Liquid chromatography-mass spectrometry, immunoblotting, and ELISA were used to screen and identify potential neovascularization-related factors in human full-thickness vascularized corneal tissues. Lipopolysaccharide was used to induce inflammation in three kinds of corneal host cells in vitro, including corneal epithelial, stromal, and endothelial cells. Fungus was used to establish an animal model of corneal neovascularization in vivo. Tube formation and spheroid sprouting assays were used to evaluate the contribution of three kinds of corneal host cells to the degree of neovascularization under various stimuli. Matrix metalloproteinase (MMP)-2, alpha-crystallin A chain (CRYAA), galectin-8, Bcl-2, neuropilin-2, MMP-9 plasmids, and recombinant human fibronectin were used to identify the key proteins of corneal host cells involved in corneal inflammatory neovascularization. FINDINGS: All three kinds of corneal host cells influenced corneal neovascularization to varying degrees. MMP-9 in human corneal epithelial cells, MMP-2, and CRYAA in human corneal stromal cells, and MMP-2 and galectin-8 in human corneal endothelial cells are potential key proteins that participate in corneal inflammatory neovascularization. INTERPRETATION: Our data indicated that both the effects of key proteins and corneal host cells involved should be considered for the treatment of corneal inflammatory neovascularization.

Cryotherapy on postoperative rehabilitation of joint arthroplasty.

PURPOSE: The effectiveness of cryotherapy on joint arthroplasty recovery remains controversial. This systematic review was conducted to assess the effectiveness of cryotherapy in patients after joint arthroplasty. METHODS: Comprehensive literature searches of several databases including Cochrane Library (2013), MEDLINE (1950-2013), and Embase (1980-2013) were performed. We sought randomised controlled trials that compared the experimental group received any form of cryotherapy with any control group after joint arthroplasty. The main outcomes were postoperative blood loss, adverse events, and pain. Analyses were performed with Revman 5.0. Results were shown as mean differences (MD) and standard deviations or as risk difference and 95 % confidence intervals (CIs). RESULTS: Ten trials comprised 660 total knee arthroplastys and three trials comprised 122 total hip arthroplastys (THAs) met the inclusion criteria. Blood loss was significantly decreased by cryotherapy (MD = -109.68; 95 % CI -210.92 to -8.44; P = 0.03). Cryotherapy did not increase the risk of adverse effect (n.s.). Cryotherapy decreased pain at the second day of postoperative (MD = -1.32; 95 % CI -2.37 to -0.27; P = 0.0003), but did not decreased pain at the first and third day of postoperative (n.s.). CONCLUSIONS: Cryotherapy appears effective in these selected patients after joint arthroplasty. The benefits of cryotherapy on blood loss after joint arthroplasty were obvious. However, the subgroup analysis indicated that cryotherapy did not decreased blood loss after THA. Cryotherapy did not increase the risk of adverse effect. Cryotherapy decreased pain at the second day of postoperative, but did not decreased pain at the first and third day of postoperative. LEVEL OF EVIDENCE: II.