High-Throughput Pre-Osteoblastic Spheroids Elevate FGF23 via PTH Signaling Pathway.

Fibroblast Growth Factor 23 (FGF23) plays a crucial role in managing renal phosphate and the synthesis of 1,25-(OH)2-vitamin D3, which is essential for bone homeostasis. Developing robust in vitro systems to study FGF23 regulating mechanisms is crucial for advancing our knowledge and identifying potential therapeutic targets. Traditional in vitro 2D culture system results in relatively low expression of FGF23, complicating further exploration of its regulatory mechanisms and potential therapeutic targets. Herein, we reported a high-throughput approach to generate pre-osteoblastic cell spheroids with enhanced FGF23 production. For this purpose, murine pre-osteoblast cell line (MC3T3-E1) were cultured in our previously reported non-adherent micro-wells (200�m in-diameter, 148�m in-depth, and 100�m space in between), and self-assembled into spheroids with a diameter of 92.3�15.0 �m after 24hrs. Compared to monolayer culture, the MC3T3-E1 spheroids showed a significant upregulation of FGF23 in both gene and protein levels after 24h serum-free induction. RNA sequencing and western blotting analysis further suggested that the enhanced FGF23 production in MC3T3-E1 spheroids was attributed to the activation of the parathyroid hormone (PTH)/PTH1R signaling pathway. Impressively, inhibition of PTH signaling through small molecular inhibitors or short hairpin RNA targeting PTH1R effectively reduced FGF23 production. In summary, the current study revealed the efficacy of the high-throughput formation of pre-osteoblast cell spheroid in stimulating FGF23 expression for mechanistic studies. Importantly, our findings highlight the potential of the current 3D spheroid system in target identification and drug discovery.

Hypergraph Learning: Methods and Practices.

Hypergraph learning is a technique for conducting learning on a hypergraph structure. In recent years, hypergraph learning has attracted increasing attention due to its flexibility and capability in modeling complex data correlation. In this paper, we first systematically review existing literature regarding hypergraph generation, including distance-based, representation-based, attribute-based, and network-based approaches. Then, we introduce the existing learning methods on a hypergraph, including transductive hypergraph learning, inductive hypergraph learning, hypergraph structure updating, and multi-modal hypergraph learning. After that, we present a tensor-based dynamic hypergraph representation and learning framework that can effectively describe high-order correlation in a hypergraph. To study the effectiveness and efficiency of hypergraph generation and learning methods, we conduct comprehensive evaluations on several typical applications, including object and action recognition, Microblog sentiment prediction, and clustering. In addition, we contribute a hypergraph learning development toolkit called THU-HyperG.

Suppression of FGF5 and FGF18 Expression by Cholesterol-Modified siRNAs Promotes Hair Growth in Mice.

FGF5 and FGF18 are key factors in the regulation of the hair follicle cycle. FGF5 is overexpressed during the late anagen phase and serves as a crucial regulatory factor that promotes the anagen-to-catagen transition in the hair follicle cycle. FGF18, which is overexpressed during the telogen phase, mainly regulates the hair follicle cycle by maintaining the telogen phase and inhibiting the entry of hair follicles into the anagen phase. The inhibition of FGF5 may prolong the anagen phase, whereas the inhibition of FGF18 may promote the transition of the hair follicles from the telogen phase to the anagen phase. In the present study, we used siRNA to suppress FGF5 or FGF18 expression as a way to inhibit the activity of these genes. Using qPCR, we showed that FGF5-targeting siRNA modified by cholesterol was more effective than the same siRNA bound to a cell-penetrating peptide at suppressing the expression of FGF5 both in vitro and in vivo. We then investigated the effects of the cholesterol-modified siRNA targeting either FGF5 or FGF18 on the hair follicle cycle in a depilated area of the skin on the back of mice. The cholesterol-modified siRNA, delivered by intradermal injection, effectively regulated the hair follicle cycle by inhibiting the expression of FGF5 and FGF18. More specifically, intradermal injection of a cholesterol-modified FGF5-targeted siRNA effectively prolonged the anagen phase of the hair follicles, whereas intradermal injection of the cholesterol-modified FGF18-targeted siRNA led to the mobilization of telogen follicles to enter the anagen phase earlier. The inhibitory effect of the cholesterol-modified FGF18-targeted siRNA on FGF18 expression was also evaluated for a topically applied siRNA. Topical application of a cream containing the cholesterol-modified FGF18-targeted siRNA on a depilated area of the skin of the back of mice revealed comparable inhibition of FGF18 expression with that observed for the same siRNA delivered by intradermal injection. These findings suggested that alopecia could be prevented and hair regrowth could be restored either through the intradermal injection of cholesterol-modified siRNA targeting FGF5 or FGF18 or the topical application of FGF18 siRNA.

Integrating Real-World Evidence in the Regulatory Decision-Making Process: A Systematic Analysis of Experiences in the US, EU, and China Using a Logic Model.

Real world evidence (RWE) and real-world data (RWD) are drawing ever-increasing attention in the pharmaceutical industry and drug regulatory authorities (DRAs) all over the world due to their paramount role in supporting drug development and regulatory decision making. However, there is little systematic documentary analysis about how RWE was integrated for the use by the DRAs in evaluating new treatment approaches and monitoring post-market safety. This study aimed to analyze and discuss the integration of RWE into regulatory decision-making process from the perspective of DRAs. Different development strategies to develop and adopt RWE by the DRAs in the US, Europe, and China were reviewed and compared, and the challenges encountered were discussed. It was found that different strategies on development of RWE were applied by FDA, EMA, and NMPA. The extent to which RWE was adopted in China was relatively limited compared to that in the US and EU, which was highly related to the national pharmaceutical environment and development stages. A better understanding of the overall goals, inputs, activities, outputs, and outcomes in developing RWE will help inform actions to harness RWD and leverage RWE for better health care decisions.

Urine Cadmium as a Risk Factor for Osteoporosis and Osteopenia: A Meta-Analysis.

Background: As society ages, the incidence of osteoporosis increases. In several studies, cadmium (Cd) is thought to be related to osteoporosis. However, there are conflicting reports about the relationship between Cd and the risk of osteoporosis and osteopenia. Therefore, the purpose of this meta-analysis was to explore the relationship between Cd and osteoporosis and osteopenia. Methods: Through a review of the literature, articles published in PubMed as of December 2020 were identified and the references of related publications and reviews were reviewed. Ultimately, 17 eligible articles were selected to determine the relationship between blood and urine Cd concentrations for the risk of osteoporosis or osteopenia. In this study, we performed a classification analysis, heterogeneity test, subgroup analysis, and evaluated publication bias. Results: A total of 17 studies were included, including seven on blood Cd and 10 on urine Cd. By combining the odds ratio (OR) and 95% confidence interval (CI) for the lowest and highest categories, the odds ratio of blood Cd concentration that increased the risk of osteoporosis or osteopenia was OR 1.21 (95% CI: 0.84-1.58) and that of urine Cd concentration that increased the risk of osteoporosis or osteopenia was OR 1.80 (95% CI: 1.42-2.18), and the results of the subgroup analysis were also consistent. Conclusions: Our research indicates that while urine cadmium (Cd) concentration may be related to increased risk of osteoporosis and osteopenia, blood Cd concentration may not. Therefore, compared to blood Cd concentration, urine Cd concentration may be more reliable as a risk factor for osteoporosis and osteopenia. This result should be interpreted with caution. Currently. research on the relationship between Cd concentration and osteoporosis and osteopenia is limited, thus, further large, high-quality prospective studies are required to elucidate the relationship between Cd concentration and osteoporosis and osteopenia.

Carnosine Stimulates Macrophage-Mediated Clearance of Senescent Skin Cells Through Activation of the AKT2 Signaling Pathway by CD36 and RAGE.

Background: Macrophages can selectively recognize and eliminate senescent cells, but this function is impaired with age, resulting in excessive accumulation of senescent cells in the skin, which ultimately causes skin aging. Therefore, enhancing the immune surveillance ability of macrophages to clear senescent keratinocytes and fibroblasts from aging skin may be an effective skin rejuvenation strategy. Methods: In this study, a macrophage and senescent skin cell co-culture model was established whereby THP-1-derived macrophages and tert-butyl hydroxide-induced senescent skin cells (HaCaT and HFF-1) were grown in the same culture. Senescent skin cells were detected by the SPiDER-ßgal assay, and the expression of secretory phenotype factors related to senescence was assayed by qPCR. The effect of carnosine on the number of SA-ß-gal positive skin cells in the macrophage-senescent skin cell co-culture was evaluated and compared with that in the senescent skin cell monoculture. Results: Carnosine promoted macrophage-mediated elimination of senescent skin cells in the co-culture. Through the AKT2 signaling pathway, carnosine upregulated the expression of CD36 and receptors for advanced glycation end products and elevated the phagocytic capacity of the macrophages, thereby promoting the ability of the macrophages to eliminate the senescent skin cells. Conclusions: Carnosine could boost the immune surveillance ability of macrophages to clear senescent keratinocytes and fibroblasts in the macrophage-senescent skin cell co-culture by activating the AKT2 signaling pathway, suggesting the possibility of using carnosine as an agent to reverse skin aging.

Direct detection of Staphylococcus aureus in positive blood cultures through molecular beacon-based fluorescence in situ hybridization.

OBJECTIVE: Clinical diagnosis of bloodstream infection diseases depends on the blood culture results. Bacterial identification by traditional methods is time-consuming. This study aimed to utilize molecular beacon-based fluorescence in situ hybridization (MB-FISH) for rapid and direct detection of Staphylococcus aureus in positive blood cultures. METHODS: Three molecular beacon probes (MB1, MB2 and MB3) were designed and synthesized to target the 16S rRNA gene fragment of S. aureus. The MB-FISH system was optimized, and the specificity of this method in detecting S. aureus was evaluated. This approach was used to test 41 g-positive clinical specimens with positive blood cultures. In addition, the consistency of this method with traditional methods was evaluated. RESULTS: Signal-to-noise ratio (S/N) of the molecular beacon MB1 was significantly higher than that of MB2 and MB3 (P < .001). The S/N ratios of MB1 probe at different concentrations were all >20. Thermal denaturation curve of the probe suggested that its hairpin structure can be opened and closed. Conditions such as deionized formamide concentration, ionic strength and temperature were optimized by monitoring the fluorescence intensity of MB1 in the presence or absence of its target sequence B1. The optimized hybridization system produced fluorescence only in S. aureus. The specificity and sensitivity of MB1 probe for detecting S. aureus in 41 specimens were 100% and 93.75%, respectively. Although sample size was small, MB-FISH appeared to be consistent with traditional culture methods (Kappa value = 0.948). CONCLUSION: MB-FISH demonstrates strong specificity and high sensitivity, and can be used for direct detection of S. aureus in positive blood cultures.

Alpha- and gamma-mangostins exhibit anti-acne activities via multiple mechanisms.

Objective: Acne is a chronic skin disease that involves four key pathogenic factors: excess sebum production, ductal epidermal hyperproliferation, Propionibacterium acnes (P. acnes) colonization, and skin inflammation. Mangostins are well-known for their anti-bacterial and anti-inflammatory effects, suggesting that mangostins may have therapeutic potential for acne. The present study aimed to explore the anti-acne effects of mangostins from the perspective of multiple pathogenic mechanisms of acne. Methods: The effects of α- and γ-mangostins on the growth of P. acnes and lipase activity were analyzed. Their effects on P. acnes-induced keratinocyte proliferation were examined by CCK-8. The expression of inflammatory genes and activation of NF-κB and MAPK signaling pathways were detected by quantitative real-time PCR and western blotting, respectively. Results: Alpha- and γ-mangostins not only inhibited the growth of P. acnes, but also reduced the proliferation of keratinocytes induced by heat-killed P. acnes. Furthermore, α- and γ-mangostins were able to suppress P. acnes-induced expression of pro-inflammatory cytokines, including TNF-α, IL-1ß, and IL-6 in keratinocytes by inhibiting the activation of NF-κB and MAPK signaling pathways. Discussion and conclusions: Mangostins appeared to possess multiple anti-acne activities, including the inhibition of P. acnes growth, regulation of keratinocytes proliferation, and attenuation of skin inflammatory reaction. Hence, mangostins might be developed into a potential therapeutic agent for the treatment of acne.

Inductive Multi-Hypergraph Learning and Its Application on View-Based 3D Object Classification.

The wide 3D applications have led to increasing amount of 3D object data, and thus effective 3D object classification technique has become an urgent requirement. One important and challenging task for 3D object classification is how to formulate the 3D data correlation and exploit it. Most of the previous works focus on learning optimal pairwise distance metric for object comparison, which may lose the global correlation among 3D objects. Recently, a transductive hypergraph learning has been investigated for classification, which can jointly explore the correlation among multiple objects, including both the labeled and unlabeled data. Although these methods have shown better performance, they are still limited due to 1) a considerable amount of testing data may not be available in practice and 2) the high computational cost to test new coming data. To handle this problem, considering the multi-modal representations of 3D objects in practice, we propose an inductive multi-hypergraph learning algorithm, which targets on learning an optimal projection for the multi-modal training data. In this method, all the training data are formulated in multi-hypergraph based on the features, and the inductive learning is conducted to learn the projection matrices and the optimal multi-hypergraph combination weights simultaneously. Different from the transductive learning on hypergraph, the high cost training process is off-line, and the testing process is very efficient for the inductive learning on hypergraph. We have conducted experiments on two 3D benchmarks, i.e., the NTU and the ModelNet40 data sets, and compared the proposed algorithm with the state-of-the-art methods and traditional transductive multi-hypergraph learning methods. Experimental results have demonstrated that the proposed method can achieve effective and efficient classification performance. We also note that the proposed method is a general framework and has the potential to be applied in other applications in practice.

Anticonvulsive and antioxidant effects of curcumin on pilocarpine-induced seizures in rats.

BACKGROUND: Curcumin, an active ingredient of turmeric with antioxidant and anti-inflammatory properties has recently been reported to have anticonvulsant effects in several animal models of epilepsy. This study aimed to investigate the effects of curcumin on the pilocarpine rat model of status epilepticus. METHODS: The effect of intraperitoneal administration of curcumin (30, 100, and 300 mg/kg) on pilocarpine-induced seizures in rats was tested. The correlation between seizure activity and hippocampal levels of nitric oxide synthase and free radicals was quantified. Whether curcumin treatment modulated these parameters was also investigated. RESULTS: Curcumin significantly increased seizure threshold at doses of 100 and 300 mg/kg. Rats with pilocarpine- induced seizures showed significantly elevated levels of malonaldehyde, nitric oxide synthase, and lactate dehydrogenase, but decreased levels of superoxide dismutase and glutathione compared with normal control rats. At doses of 100 and 300 mg/kg, curcumin reversed the effects of pilocarpine-induced seizures on nitric oxide synthase, lactate dehydrogenase, glutathione, and superoxide dismutase. However, curcumin did not restore the elevated malonaldehyde levels. CONCLUSION: Curcumin has anticonvulsant activity in the pilocarpine rat model of seizures, and that modulation of free radicals and nitric oxide synthase may be involved in this effect.

Deficits in spatial learning and memory is associated with hippocampal volume loss in aged apolipoprotein E4 mice.

Apolipoprotein E ε4 (ApoE4) has been implicated as a potential genetic risk factor for dementia. In this study, we investigate the effect of ApoE4 on learning and memory, changes in brain volume and neuroinflammatory responses in brain of ApoE4 transgenic mice. Four groups of male mice with ApoE4 and age-matched wild type (WT) (6-, 12-, 18- and 24-month) were studied. Spatial learning and retaining of mice was examined in the Morris Water Maze (MWM). Changes in brain volume (including the whole brain, hippocampus, cortex, total ventricles, and caudate putamen) were assessed by using 7T small animal MRI. Neuroinflammatory responses were analyzed by measuring the levels of microglia (Iba-1), iNOS, TNFα, and IL-6 quantitatively. In the MWM, ApoE4 mice showed longer escape latency (p < 0.05) and swim distance (p < 0.05) at age 12 month and older, comparing with the WT mice. They also demonstrated poor memory retention in the probe test (p < 0.05). Brain atrophy was significant in ApoE4 mice than age-matched WT mice (18 months: 0.079 ± 0.004 versus 0.086 ± 0.003, p = 0.018; and 24 months: 0.074 ± 0.005 versus 0.084 ± 0.006, p = 0.008). The expression of Iba-1, iNOS, and TNFα in hippocampus and cortex were significantly higher in ApoE4 mice than in WT mice at 12 months and older. These data suggest that ApoE4 plays an important role in learning and memory impairment. These deficits are associated with neuroinflammatory responses that may in turn lead to atrophy in hippocampus and cortex.

Centrally administered pertussis toxin inhibits microglia migration to the spinal cord and prevents dissemination of disease in an EAE mouse model.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) models are important vehicles for studying the effect of infectious elements such as Pertussis toxin (PTx) on disease processes related to acute demyelinating encephalomyelitis (ADEM) or multiple sclerosis (MS). PTx has pleotropic effects on the immune system. This study was designed to investigate the effects of PTx administered intracerebroventricularly (icv) in preventing downstream immune cell infiltration and demyelination of the spinal cord. METHODS AND FINDINGS: EAE was induced in C57BL/6 mice with MOG(35-55). PTx icv at seven days post MOG immunization resulted in mitigation of clinical motor symptoms, minimal T cell infiltration, and the marked absence of axonal loss and demyelination of the spinal cord. Integrity of the blood brain barrier was compromised in the brain whereas spinal cord BBB integrity remained intact. PTx icv markedly increased microglia numbers in the brain preventing their migration to the spinal cord. An in vitro transwell study demonstrated that PTx inhibited migration of microglia. CONCLUSION: Centrally administered PTx abrogated migration of microglia in EAE mice, limiting the inflammatory cytokine milieu to the brain and prevented dissemination of demyelination. The effects of PTx icv warrants further investigation and provides an attractive template for further study regarding the pleotropic effects of infectious elements such as PTx in the pathogenesis of autoimmune disorders.

Curcumin inhibits amygdaloid kindled seizures in rats.

BACKGROUND: Curcumin can reduce the severity of seizures induced by kainate acid (KA), but the role of curcumin in amygdaloid kindled models is still unknown. This study aimed to explore the effect of curcumin on the development of kindling in amygdaloid kindled rats. METHODS: With an amygdaloid kindled Sprague-Dawley (SD) rat model and an electrophysiological method, different doses of curcumin (10 mgxkg(-1)xd(-1) and 30 mgxkg(-1)xd(-1) as low dose groups, 100 mgxkg(-1)xd(-1) and 300 mgxkg(-1)xd(-1) as high dose groups) were administrated intraperitoneally during the whole kindling days, by comparison with the course of kindling, afterdischarge (AD) thresholds and the number of ADs to reach the stages of class I to V seizures in the rats between control and experimental groups. One-way or two-way ANOVA and Fisher's least significant difference post hoc test were used for statistical analyses. RESULTS: Curcumin (both 100 mgxkg(-1)xd(-1) and 300 mgxkg(-1)xd(-1)) significantly inhibited the behavioral seizure development in the (19.80 +/- 2.25) and (21.70 +/- 2.21) stimulations respectively required to reach the kindled state. Rats treated with 100 mgxkg(-1)xd(-1) curcumin 30 minutes before kindling stimulation showed an obvious increase in the stimulation current intensity required to evoke AD from (703.3 +/- 85.9) microA to (960.0 +/- 116.5) microA during the progression to class V seizures. Rats treated with 300 mgxkg(-1)xd(-1) curcumin showed a significant increase in the stimulation current intensity required to evoke AD from (735.0 +/- 65.2) microA to (867.0 +/- 93.4) microA during the progression to class V seizures. Rats treated with 300 mgxkg(-1)xd(-1) curcumin required much more evoked ADs to reach the stage of class both IV (as (199.83 +/- 12.47) seconds) and V seizures (as (210.66 +/- 10.68) seconds). Rats treated with 100 mgxkg(-1)xd(-1) curcumin required much more evoked ADs to reach the stage of class V seizures (as (219.56 +/- 18.24) seconds). CONCLUSION: Our study suggests that curcumin has a potential antiepileptogenic effect on kindling-induced epileptogenesis.

APOE 4 polymorphism results in early cognitive deficits in an EAE model.

Recent clinical studies have identified an association between APOE 4 and cognitive deficits in patients with multiple sclerosis. We induced experimental autoimmune encephalomyelitis (EAE) in APOE knockout (KO) and human APOE 4 knockin (E4) mice to study the interaction of APOE and neuroinflammation on cognition. After EAE induction, KO and E4 showed significant deficits in spatial learning and recall. Regional decreases in choline acetyltransferase localized to the hippocampus. Induction of EAE in a transgenic APOE animal provides a template from which we can decipher the role APOE has on cognition in the context of neuroinflammation.