Parsing the effect of co-culture with brain organoids on Diffuse Intrinsic Pontine Glioma (DIPG) using quantitative proteomics.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly brain cancers in children for which there is no effective treatment. This can partly be attributed to preclinical models that lack essential elements of the in vivo tissue environment, resulting in treatments that appear promising preclinically, but fail to result in effective cures. Recently developed co-culture models combining stem cell-derived brain organoids with brain cancer cells provide tissue dimensionality and a human-relevant tissue-like microenvironment. As these models are technically challenging, we aimed to establish whether interaction with the organoid influences DIPG biology and thus warrants their use. To address this question DIPG24 cells were cultured with pluripotent stem cell-derived cortical organoids. We created "mosaic" co-cultures enriched for tumour cell-neuronal cell interactions versus "assembloid" co-cultures enriched for tumour cell-tumour cell interactions. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to analyse the proteomes of DIPG fractions isolated by flow-assisted cell sorting. Control proteomes from DIPG spheroids were compared with DIPG cells isolated from mosaic and assembloid co-cultures. This suggested changes in cell interaction with the external environment reflected by decreased gene ontology terms associated with adhesion and extracellular matrix, and increased DNA synthesis and replication, in DIPG24 cells under either co-culture condition. By contrast, the mosaic co-culture was associated with neuron-specific brahma-associated factor (nBAF) complex signalling, a process associated with neuronal maturation. We propose that co-culture with brain organoids is a valuable tool to parse the contribution of the brain microenvironment to DIPG tumour biology.

Protective effects of polydatin against bone and joint disorders: the in vitro and in vivo evidence so far.

Polydatin is an active polyphenol displaying multifaceted benefits. Recently, growing studies have noticed its potential therapeutic effects on bone and joint disorders (BJDs). Therefore, this article reviews recent in vivo and in vitro progress on the protective role of polydatin against BJDs. An insight into the underlying mechanisms is also presented. It was found that polydatin could promote osteogenesis in vitro, and symptom improvements have been disclosed with animal models of osteoporosis, osteosarcoma, osteoarthritis and rheumatic arthritis. These beneficial effects obtained in laboratory could be mainly attributed to the bone metabolism-regulating, anti-inflammatory, antioxidative, apoptosis-regulating and autophagy-regulating functions of polydatin. However, studies on human subjects with BJDs that can lead to early identification of the clinical efficacy and adverse effects of polydatin have not been reported yet. Accordingly, this review serves as a starting point for pursuing clinical trials. Additionally, future emphasis should also be devoted to the low bioavailability and prompt metabolism nature of polydatin. In summary, well-designed clinical trials of polydatin in patients with BJD are in demand, and its pharmacokinetic nature must be taken into account.

Preparation and Biocompatibility of Core-Shell Microspheres for Sequential, Sustained Release of BMP-2 and VEGF.

Bone defect repair remains a challenge in orthopedics. This study describes the development and potential effectiveness of vascular endothelial growth factor (VEGF)/bone morphogenetic protein-2 (BMP-2) shell-core microspheres for promoting bone regeneration. Poly(L-lactic acid)/polylactic-co-glycolic acid (PLLA/PLGA) core-shell microspheres loaded with VEGF and BMP-2 were prepared by a coaxial electrospray technique, and their surface morphology, core-shell distribution, and particle size were examined. Different groups of microspheres were prepared with different placement of the growth factors, and the encapsulation efficiency and in vitro release curves were measured. Additionally, the effects of the different groups of microspheres on the proliferation and differentiation of osteoblasts and vascular endothelial cells were investigated. The prepared microspheres had a core-shell structure with good homogeneity and dispersion, a clear boundary, and a smooth surface. On scanning electron microscopy, the mean diameter of the microspheres was similar for all six preparations (P > 0.05). During in vitro release, growth factor was initially released via a brief burst release from the outer shell of the microsphere followed by a slower sustained release. The release of growth factors from the inner core remained relatively slow and sustained. Sequential release of different growth factors was achieved through the inconsistent release rates from the microsphere shell and inner core. All groups of microspheres showed no cytotoxicity, good biocompatibility, and the ability to promote osteoblast proliferation. The microspheres loaded with BMP-2 also promoted osteoblast differentiation, and VEGF-loaded microspheres promoted the proliferation and differentiation of vascular endothelial cells. The BMP-2 (core)/VEGF (shell) microsphere group best promoted osteoblast differentiation. The microspheres prepared in this study exhibited slow sequential release of BMP-2 and VEGF and showed good biocompatibility along with the ability to promote osteoblast differentiation and vascular endothelial cell proliferation.

Extracellular vesicles from human umbilical cord mesenchymal stem cells reduce lipopolysaccharide-induced spinal cord injury neuronal apoptosis by mediating miR-29b-3p/PTEN.

OBJECTIVE: This study investigated the molecular mechanism of whether hUC-MSCs-EVs repressed PTEN expression and activated the PI3K/AKT pathway through miR-29b-3p, thus inhibiting LPS-induced neuronal injury. METHODS: hUC-MSCs were cultured and then identified. Cell morphology was observed. Alizarin red, oil red O, and alcian blue staining were used for inducing osteogenesis, adipogenesis, and chondrogenesis. EVs were extracted from hUC-MSCs and identified by transmission electron microscope observation and Western blot. SCI neuron model was established by 24h lipopolysaccharide (LPS) induction. After the cells were cultured with EVs without any treatment, uptake of EVs by SCI neurons, miR-29b-3p expression, cell viability, apoptosis, caspase-3, cleaved caspase-3, caspase 9, Bcl-2, PTEN, PI3K, AKT, and p-Akt protein levels, caspase 3 and caspase 9 activities, and inflammatory factors IL-6 and IL-1ß levels were detected by immunofluorescence labeling, RT-qPCR, MTT, flow cytometry, Western blot, caspase 3 and caspase 9 activity detection kits, and ELISA. The binding sites between PTEN and miR-29b-3p were predicted by the database and verified by dual-luciferase assay. RESULTS: LPS-induced SCI cell model was successfully established, and hUC-MSCs-EVs inhibited LPS-induced apoptosis of injured spinal cord neurons. EVs transferred miR-29b-3p into LPS-induced injured neurons. miR-29b-3p silencing reversed EV effects on reducing LPS-induced neuronal apoptosis. miR-29b-3p reduced LPS-induced neuronal apoptosis by targeting PTEN. After EVs-miR-inhi and si-PTEN treatment, inhibition of the PI3K/AKT pathway reversed hUC-MSCs-EVs effects on reducing LPS-induced neuronal apoptosis. CONCLUSION: hUC-MSCs-EVs activated the PI3K/AKT pathway by carrying miR-29b-3p into SCI neurons and silencing PTEN, thus reducing neuronal apoptosis.

Macrophage responses to selective laser-melted Ti-6Al-4V scaffolds of different pore geometries and the corresponding osteoimmunomodulatory effects toward osteogenesis.

Following recent advances in osteoimmunology, there is growing recognition of the vital role of immune cells in the osteogenesis process. The 3D-printed scaffold, as a substitute for injured and/or diseased bone tissues, has demonstrated satisfactory pro-osteogenetic performance. However, whether immune cells prompt the above pro-osteogenetic performance has not been elucidated in detail. In the present study, highly controllable Ti-6Al-4V scaffolds with different pore geometries were fabricated using a selective laser-melting technique, to reveal their osteoimmunological functions with macrophages. The results showed that macrophages displayed characteristics of M2 phenotype in response to scaffolds. As a result, an anti-inflammatory microenvironment was generated. When the pore geometry was considered, such observations were more apparent with the hexagonal pore scaffold than with the triangular one. In addition, inhibition of the toll-like receptor signaling pathway in macrophages has been proposed to cause the above phenomenon. Upon applying conditioned media derived from macrophages on pre-osteoblasts, the hexagonal pore scaffold group was found to significantly enhance osteoblastic differentiation, via macrophage-to-implant interactions. However, the effect of triangular pore scaffold was not statistically significant compared to that of hexagonal pore scaffolds or nonporous samples. In an attempt to quantify scaffold pore geometries, it was suggested that pores with higher circularity values tended to induce M2 polarization of macrophages, promote an anti-inflammatory milieu, and therefore, achieve better osteogenetic performance via immunomodulation.

Clinicopathological characteristics and prognosis of hepatitis B associated membranous nephropathy and idiopathic membranous nephropathy complicated with hepatitis B virus infection.

The main objective of this study is to analyze the clinical and pathological features and prognosis of patients with Hepatitis B associated membranous nephropathy (HBV-MN) and idiopathic membranous nephropathy (IMN) complicated with hepatitis B virus (HBV) infection. This study will provide more basis for diagnosis and prognosis evaluation. A total of 50 patients with HBV-MN were included in this study. 56 IMN patients complicated with HBV infection diagnosed during the same period formed the control group. Parameters including blood routine, urine routine and plasma levels of albumin (ALB), serum creatinine (SCR), blood urea nitrogen (BUN), urea acid (UA), total cholesterol (T-CHO), triglycerides (TG), complement C3 and C4, glutamic pyruvic transaminase (ALT), glutamic pyruvic transaminase (AST), 24-h urinary protein quantification (24 h-TP), renal phospholipase A2 receptor (PLA2R) and HBV related markers during the hospitalization and outpatient follow-up study period were collected for all the patients. The proportion of male patients was high in both groups. The average age of the HBV-MN group was 37.2 ± 14.187 years old, it was younger compared with the IMN group (P = 0.003). Nephrotic syndrome was the major clinical manifestation among patients. There was no significant difference between the two groups in the levels of anemia, microscopic hematuria, renal dysfunction, liver dysfunction, liver cirrhosis. The level of serum C3 and C4 in the HBV-MN group was lower compared with the IMN group (P = 0.002, P = 0.014). In the HBV-MN group, serum HBV markers were negative in 6 (12%) patients, 4 patients (8%) were positive for PLA2R in serum, and 5 patients (10%) were positive for PLA2R in renal tissue. Stronger IgG1 and C1q and weaker IgG4 staining were found in HBV-MN group renal tissues (P = 0.003, P = 0.025, and P = 0.001, respectively). There were no statistical differences compared with serum and renal PLA2R between HBV-MN and IMN groups (P = 0.098, P = 0.109). During the 1-year follow-up, there was no significant difference in complete remission rate between the two groups (P = 0.7739). Renal biopsy is crucial to diagnose HBV-MN. IgG subtypes in the HBV-MN group were mainly IgG1 deposition, while those in IMN complicated with HBV infection group were mainly IgG4 deposition. When HBV-associated antigen and PLA2R are present in renal tissue, lower level of serum C3 and C4, high intensity of renal C1q and IgG1 is more supportive of HBV-MN. The positive of PLA2R in serum and renal tissue in differentiating HBV from IMN complicated with HBV infection remains to be discussed.

Medium-Term Follow-Up Outcomes of One-Stage Posterior Lumbosacral or Lumbopelvic Fixation in the Management of Lumbosacral Junction Tuberculosis in Adults.

OBJECTIVE: To evaluate the medium-term outcomes of one-stage posterior lumbosacral or lumbopelvic fixation treatment of lumbosacral junction tuberculosis in adults. METHODS: This retrospective study enrolled a total of 38 adult patients (24 males and 14 females) with an average age of 48.0 ± 13.0 years (range, 25-75 years) during the period from February 2008 to July 2015. All patients were treated by one-stage posterior debridement, interbody fusion, lumbosacral or lumbopelvic fixation, and postural drainage. After pedicle screw or iliac screw fixation, a hemi-laminectomy or laminectomy was performed on the severely damaged side of the lesion segment. Intervertebral bone grafting and intertransverse bone grafting were performed after clearing the focus of tuberculosis. All cases were followed up for at least 5 years. Intraoperative blood loss, operative time, erythrocyte sedimentation rate (ESR), pain intensity was assessed by visual analog scale (VAS) score; neurological function was assessed by Japanese Orthopaedic Association (JOA) score; quality of life was assessed by Oswestry Disability Index (ODI); functional outcome, lumbosacral angle, and fusion time were gathered and analyzed. All data expressed as mean ± standard deviation. RESULTS: During the 66.2 ± 4.4 months (range, 60-78 months) follow-up, all patients achieved clinical cure without severe complications. The intraoperative blood loss was 726.3 ± 151.9 mL (range, 400-1100 mL) and the operative time was 137.6 ± 22.5 min (range, 110-200 min). The ESR decreased to normal levels within (11.8 ± 2.6 mm/h) 3 months postoperatively. The VAS score significantly decreased from 6.8 ± 1.1 preoperatively to 0.8 ± 0.7 at the final follow-up (P < 0.01). The mean JOA improved from preoperative 18.5 ± 2.9 to 26.9 ± 1.1 at the last visit (P < 0.01). The mean ODI was 44.3 ± 6.7 and significantly decreased to 9.3 ± 1.9 at the final observation (P < 0.01). Patient-reported outcomes as measured by Kirkaldy-Willis criteria were excellent in 21 cases, good in 16 cases, and fair in one case; there were no poor outcomes. Lumbosacral angle increased from the preoperative values of 21.7° ± 1.8° to the postoperative values of 26.4° ± 1.4° (P < 0.01), with an angle loss of 1.2° ± 0.7° at the last follow-up. Bone fusion occurred on average 12.8 ± 1.9 months (range, 9-15 months) after surgery. No nonunion, pseudarthrosis, loosening or fracture of instruments occurred at the last follow-up. CONCLUSION: One-stage posterior debridement, interbody fusion, lumbosacral or lumbopelvic fixation, and postural drainage according to the severity of sacral destruction is an effective and highly safe procedure to treat lumbosacral junction tuberculosis in adults.

Presymptomatic Dutch-Type Hereditary Cerebral Amyloid Angiopathy-Related Blood Metabolite Alterations.

BACKGROUND: Cerebral amyloid angiopathy (CAA) is one of the major causes of intracerebral hemorrhage and vascular dementia in older adults. Early diagnosis will provide clinicians with an opportunity to intervene early with suitable strategies, highlighting the importance of pre-symptomatic CAA biomarkers. OBJECTIVE: Investigation of pre-symptomatic CAA related blood metabolite alterations in Dutch-type hereditary CAA mutation carriers (D-CAA MCs). METHODS: Plasma metabolites were measured using mass-spectrometry (AbsoluteIDQ® p400 HR kit) and were compared between pre-symptomatic D-CAA MCs (n = 9) and non-carriers (D-CAA NCs, n = 8) from the same pedigree. Metabolites that survived correction for multiple comparisons were further compared between D-CAA MCs and additional control groups (cognitively unimpaired adults). RESULTS: 275 metabolites were measured in the plasma, 22 of which were observed to be significantly lower in theD-CAAMCs compared to D-CAA NCs, following adjustment for potential confounding factors age, sex, and APOE ε4 (p < 00.05). After adjusting for multiple comparisons, only spermidine remained significantly lower in theD-CAAMCscompared to theD-CAA NCs (p  < 0.00018). Plasma spermidine was also significantly lower in D-CAA MCs compared to the cognitively unimpaired young adult and older adult groups (p < 0.01). Spermidinewas also observed to correlate with CSF Aß40 (rs = 0.621, p = 0.024), CSF Aß42 (rs = 0.714, p = 0.006), and brain Aß load (rs = -0.527, p = 0.030). CONCLUSION: The current study provides pilot data on D-CAA linked metabolite signals, that also associated with Aß neuropathology and are involved in several biological pathways that have previously been linked to neurodegeneration and dementia.

Proteome analysis of endometrial tissue from patients with PCOS reveals proteins predicted to impact the disease.

Polycystic ovary syndrome (PCOS) is a complex disease that causes an ovulatory infertility in approximately 10% of reproductive-age women. We searched for candidate proteins that might contribute to endometrial receptivity defects in PCOS patients, and result in adverse reproductive outcomes. Shotgun proteomics approach was used to investigate the proteome profile of the endometrium at the luteal phase in PCOS patients compared to healthy fertile individuals. Biological process and pathway analyses were conducted to categorize the proteins with differential expressions. Confirmation was performed for a number of proteins via immunoblotting in new samples. 150 proteins with higher abundance, and 46 proteins with lower abundance were identified in the endometrial tissue from PCOS patients compared to healthy fertile individuals. The proteins with higher abundance were enriched in protein degradation, cell cycle, and signaling cascades. Proteins with lower abundance in PCOS patients were enriched in extracellular matrix (ECM) composition and function, as well as the salvage pathway of purine biosynthesis. Metabolism was the most affected biological process with over 100 up-regulated, and approximately 30 down-regulated proteins. Our results indicate significant imbalances in metabolism, proteasome, cell cycle, ECM related proteins, and signaling cascades in endometrial tissue of PCOS, which may contribute to poor reproductive outcomes in these patients. We postulate that the endometria in PCOS patients may not be well-differentiated and synchronized for implantation. Possible roles of the above-mentioned pathways that underlie implantation failure in PCOS will be discussed. Our findings need to be confirmed in larger populations.

Amyloid-beta peptide neurotoxicity in human neuronal cells is associated with modulation of insulin-like growth factor transport, lysosomal machinery and extracellular matrix receptor interactions.

Extracellular deposits of the amyloid-beta peptide (Aß) are known as the main pathological hallmark of Alzheimer's disease. In Alzheimer's disease, neurons are injured and die throughout the brain, a process in which Aß neurotoxicity is considered to play an important role. However, the molecular mechanisms underlying Aß toxicity that lead to neurodegeneration are not clearly established. Here we have elucidated the molecular pathways and networks which are impacted by Aß in neurons using SH-SY5Y human neuroblastoma cells as a model. These cells were treated with Aß1-42 peptides to study changes in biochemical networks using tandem mass tag labeled quantitative proteomic technique followed by computational analysis of the data. The molecular impacts of Aß on cells were evident in a time- and dose-dependent manner, albeit the duration of treatment induced greater differential changes in cellular proteome compared to the effects of concentration. Aß induced early changes in proteins associated with lysosomes, collagen chain trimerization and extracellular matrix receptor interaction, complement and coagulation cascade, oxidative stress induced senescence, ribosome biogenesis, regulation of insulin-like growth factor transport and uptake by insulin-like growth factor-binding protein. These novel findings provide molecular insights on the effects of Aß on neurons, with implications for better understanding the impacts of Aß on early neurodegeneration in Alzheimer's disease pathology.

Case Report: Glucocorticoids Combined With Immunosuppressant in the Treatment of Acromegaly Complicated With Focal Segmental Glomerulosclerosis.

Background: Acromegaly is a chronic disease caused by excessive secretion of growth hormone (GH), which circulates and stimulates the liver and body tissues to produce insulin-like growth factor type 1 (IGF-1). Experimental studies have shown that excessive secretion of GH is related to glomerular sclerosis, and elevated IGF-1 levels may be involved in the occurrence of glomerular hypertrophy. But relevant clinical cases are rare. Here, we reported a case of acromegaly complicated with focal segmental glomerulosclerosis (FSGS). Case Presentation: A 49-year-old man was admitted to our hospital because of acromegaly for more than 10 years and proteinuria for more than 3 years. Acromegaly was confirmed by contrast-enhanced magnetic resonance imaging, minimally invasive surgery and pathology. The results of renal biopsy showed FSGS-NOS (not otherwise specified) with ischemic renal injury and mesangial IgA deposition. One month after transnasal transsphenoidal space occupying resection, GH and urinary protein decreased significantly, and nephropathy was partially relieved. In the next 4 months, GH stabilized at the normal level, while urinary protein gradually increased. When the urinary protein increased to 4.2 g/d, the dosage of glucocorticoids increased to 20 mg/d, and tacrolimus 1 mg/d were added, and the urinary protein decreased again. However, when the urinary protein decreased to 0.43 g/d, the patient stopped taking glucocorticoids and tacrolimus, and the urinary protein increased to 2.85 g/d after 8 months, but the GH was still in the normal range. Conclusion: In this case, GH is partially involved in the formation of FSGS. Not only does surgery reduce the effects of GH, but low doses of glucocorticoids and immunosuppressant are effective in slowing the progression of kidney disease, at least in reducing urinary protein.

Clinical Characteristics of 1378 Inpatients with Spinal Tuberculosis in General Hospitals in South-Central China.

In this retrospective study, charts of inpatients with spinal tuberculosis (STB) treated in large-scale general hospitals in Changsha, Hunan, China, between 2007 and 2016 were reviewed to investigate their clinical characteristics. Demographic, epidemiological and clinical features, imaging findings, treatment methods, and prognosis were summarized and analyzed. There were 1378 patients, 805 males and 573 females, with a mean age of 43.7 years. The mean interval between symptom onset and diagnosis was 16.0 months (range 15 days-240 months). The incidence of back pain, radicular pain and symptoms of systemic toxicity was 92.5%, 40.1%, and 32.1%, respectively. The rate of neurological impairment was 49.9 %. STB was present in two or more vertebrae in 91.1% of patients, with two adjacent vertebrae being involved in 67.9% of them. The lumbar segment (38.2%) was the most frequently affected, followed by the thoracic spine (35.7%). The sacrococcygeal area was the least frequently involved (0.8%). Abscesses were detected in 65.5% of patients. One thousand patients (72.6%) were managed with surgery and 378 (27.4%) with anti-TB drugs only. Cure was achieved in 1215 patients (88.2%), whereas 49 (3.5 %) had relapses. Concomitant pulmonary TB (PTB) was diagnosed in 366 patients (26.6%) and 63 (4.6%) had concomitant diabetes. Compared with the previous five years, the number of older patients, urban patients, and medical staff with STB had increased by 6.1%, 5.2%, and 1.3%, respectively in the five years studied. STB remains a severe public health problem that cannot be ignored. Most of the patients ignored early symptoms and therefore received untimely treatment. Thus, surveillance for and treatment of STB in South-central China requires strengthening. In addition to the current China-wide database of patients with PTB, a China-wide database of patients with STB should also be set up.

Loss of Shp2 Rescues BDNF/TrkB Signaling and Contributes to Improved Retinal Ganglion Cell Neuroprotection.

Glaucoma is characterized by the loss of retinal ganglion cells (RGC), and accordingly the preservation of RGCs and their axons has recently attracted significant attention to improve therapeutic outcomes in the disease. Here, we report that Src homology region 2-containing protein tyrosine phosphatase 2 (Shp2) undergoes activation in the RGCs, in animal model of glaucoma as well as in the human glaucoma tissues and that Shp2 dephosphorylates tropomyosin receptor kinase B (TrkB) receptor, leading to reduced BDNF/TrkB neuroprotective survival signaling. This was elucidated by specifically modulating Shp2 expression in the RGCs in vivo, using adeno-associated virus serotype 2 (AAV2) constructs. Shp2 upregulation promoted endoplasmic reticulum (ER) stress and apoptosis, along with functional and structural deficits in the inner retina. In contrast, loss of Shp2 decelerated the loss of RGCs, preserved their function, and suppressed ER stress and apoptosis in glaucoma. This report constitutes the first identification of Shp2-mediated TrkB regulatory mechanisms in the RGCs that can become a potential therapeutic target in both glaucoma and other neurodegenerative disorders.

Clinically Relevant Post-Translational Modification Analyses-Maturing Workflows and Bioinformatics Tools.

Post-translational modifications (PTMs) can occur soon after translation or at any stage in the lifecycle of a given protein, and they may help regulate protein folding, stability, cellular localisation, activity, or the interactions proteins have with other proteins or biomolecular species. PTMs are crucial to our functional understanding of biology, and new quantitative mass spectrometry (MS) and bioinformatics workflows are maturing both in labelled multiplexed and label-free techniques, offering increasing coverage and new opportunities to study human health and disease. Techniques such as Data Independent Acquisition (DIA) are emerging as promising approaches due to their re-mining capability. Many bioinformatics tools have been developed to support the analysis of PTMs by mass spectrometry, from prediction and identifying PTM site assignment, open searches enabling better mining of unassigned mass spectra-many of which likely harbour PTMs-through to understanding PTM associations and interactions. The remaining challenge lies in extracting functional information from clinically relevant PTM studies. This review focuses on canvassing the options and progress of PTM analysis for large quantitative studies, from choosing the platform, through to data analysis, with an emphasis on clinically relevant samples such as plasma and other body fluids, and well-established tools and options for data interpretation.