Designing an ideal 3D-bioprint conduit for axonal repair and regeneration: a neurosurgical perspective

Peripheral nerve injuries occur through three mechanisms, specifically, crush, compression or transection. Disruption of communication between the peripheral and central nervous system follows and leads to motor and sensory deficits. Peripheral nerves in humans have a limited capacity to self-regenerate following injury, which makes nerve transfer the current gold-standard for treatment. Functional nerve regeneration is contingent on several factors ranging from span of injury and the age of the patient. Bioprinted nerve guidance conduits are an emerging candidate for treating peripheral nerve injuries. To optimize the performance of nerve guidance conduits, a firm understanding of neurobiology and the pathophysiology following injury is necessary. This article provides an overview of nerve regeneration and the desirable features when designing a nerve conduit from a neurosurgical perspective

Machine intelligence for nerve conduit design and production

Nerve guidance conduits (NGCs) have emerged from recent advances within tissue engineering as a promising alternative to autografts for peripheral nerve repair. NGCs are tubular structures with engineered biomaterials, which guide axonal regeneration from the injured proximal nerve to the distal stump. NGC design can synergistically combine multiple properties to enhance proliferation of stem and neuronal cells, improve nerve migration, attenuate inflammation and reduce scar tissue formation. The aim of most laboratories fabricating NGCs is the development of an automated process that incorporates patient-specific features and complex tissue blueprints (e.g. neurovascular conduit) that serve as the basis for more complicated muscular and skin grafts. One of the major limitations for tissue engineering is lack of guidance for generating tissue blueprints and the absence of streamlined manufacturing processes. With the rapid expansion of machine intelligence, high dimensional image analysis, and computational scaffold design, optimized tissue templates for 3D bioprinting (3DBP) are feasible. In this review, we examine the translational challenges to peripheral nerve regeneration and where machine intelligence can innovate bottlenecks in neural tissue engineering

Identification of clinically useful plasma miRNA as minimally invasive biomarkers for early stage Non-Small Cell Lung Carcinoma (NSCLC)

Lung cancer is currently the leading cause of cancer death in both developing and developed countries. Given that lung cancer has poor prognosis in later stages, it is essential to achieve an early diagnosis to maximise a patient’s overall survival. Lung adenocarcinoma (LUAD), a subtype of non-small cell lung cancer (NSCLC), has the highest incidence in both smokers and non-smokers. Therefore, there is an urgent need to develop methods to provide both sensitive and specific lung cancer diagnosis.The current standard and non-invasive screening method, low‐dose computed tomography (LDCT), is the only radiological method which shows to have mortality benefits from multiple large randomized clinical trials (RCT). However, these RCTs also found LDCT to have a significant false positive rate of 96% which resulted in unnecessary invasive biopsies being performed. Due to the lack of sensitive and specific screening methods for the early detection of lung cancer, there is an urgent need for alternative biomarkers that are minimally invasive and may provide diagnostic, and/or prognostic potential. Circulating biomarkers can be readily detectable in blood and have been extensively studied as prognosis markers. Circulating microRNA (miRNA) been investigated for these purposes as an augmentation to LDCT, or as direct diagnosis of lung cancer. There is however, a lack of consensus across the studies on what miRNAs are the most clinically useful. \ua0\ua0In steps to address this problem, we performed a comprehensive unbiased assessment of all known miRNAs in a progression series of 25 human LUAD tissues. In the microarray data, we identified four novel findings. Firstly, we identified 13 miRNAs with differential expression changes across all four stages of LUAD tissue. Secondly, these 13 miRNAs include miR-4454, miR-4634 miR-4707-5p, and miR-4492, which have not been reported in previous literature related to early LUAD. Thirdly, three of the 13 miRNAs: miR-96-5p, miR-182-5p, and miR-183-5p also show a strong correlation between LUAD disease progression and expression level. Finally, we identified miR-31-5p as the strongest correlation with LUAD stage progression out of all the miRNAs investigated in our dataset.We validated the microarray expression of miR-31-5p, miR-96-5p, miR-182-5p, and miR-183-5p expression level by quantitative real time polymerase chain reaction (qRT-PCR) in the 25 LUAD tissues present on the array. Next, to confirm that miRNA expression may be related early stage LUAD, we obtained an independent cohort of stage I LUAD (n=23) and 11 adjacent normal tissues. Real-time qPCR confirmed that all four miRNAs’ expression level were significantly upregulated (P≤0.05) in stage I LUAD with miR-31-5p increasing by ~35-fold, miR-96-5p by ~8-fold, miR-182-5p by ~5-fold, and miR-183-5p by ~6-fold.Next, to explore the notion that the miRNA panel may serve as circulating biomarkers for early detection and/or prognosis, we obtained plasma samples from 56 patients (32 Stage I, 5 Stage II, 8 Stage III, and 5 Stage IV) with LUAD along with six healthy control samples (this set includes 26 samples which had a matched \ua0tissue sample). As previously observed in stage I tissue, the circulating expression levels of miR-31-5p, miR-182-5p, and miR-183-5p were significantly increased. Interestingly, miR-96-5p expression was only detected in three out of 56 plasma samples and was deemed not to be a robust biomarker. As such, only the diagnostic utility of miR-31-5p, miR-182-5p, and miR-183-5p were investigated further. The receiver operator curve (ROC) area under the curve (AUC) of miR-31-5p, miR-182-5p, miR-183-5p ranged from 0.8750 - 0.8229 when stage I was compared with normal controls. Similar AUC scores were observed when stage II tissues were investigated. Therefore, these three miRNAs may have diagnostic utility for early detection of LUAD in plasma which warrants further investigation.In summary, we have identified miR-4454, miR-4707-5p, miR-4492, and miR-4634 as novel miRNA related to LUAD from the microarray. Moreover, miR-31-5p, miR-96-5p, miR-182-5p, and miR-183-5p tissue expression level have a strong correlation with LUAD disease progression. Finally, plasma miR-31-5p, miR-182-5p, and miR-183-5p have non-invasive diagnostic utility individually in early staged LUAD

High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway

Long-chain acyl-CoA synthetases (ACSL) play key roles in fatty acid metabolism in liver and other metabolic tissues in an isozyme-specific manner. In this study, we examined the effects of a fructose-enriched diet on expressions of ACSL isoforms in the liver of hamsters. We showed that the fructose diet markedly reduced the mRNA and protein expressions of ACSL3 in hamster liver without significant effects on other ACSLs. The decrease in ACSL3 abundance was accompanied by a reduction in ACSL-catalyzed synthesis of arachidonyl-CoA and oleoyl-CoA in liver homogenates of hamsters fed the fructose diet as opposed to normal diet. We further showed that fructose diet specifically reduced expressions of three key components of the LXR signaling pathway, namely, liver X receptor (LXR)alpha, LXR beta, and retinoid X receptor (RXR)beta. Exogenous expression and activation of LXR alpha/beta increased hamster ACSL3 promoter activities in a LXR-responsive element (LXRE)-dependent fashion. Finally, we showed that treating hamsters with LXR agonist GW3965 increased hepatic ACSL3 expression without affecting other ACSL isoforms. Furthermore, the ligand-induced increases of ACSL3 expression were accompanied with the reduction of hepatic triglyceride levels in GW3965-treated hamster liver. Altogether, our studies demonstrate that fructose diet has a negative impact on LXR signaling pathway in liver tissue and reduction of ACSL3 expression/activity could be a causal factor for fructose-induced hepatic steatosis.-Dong, B., C. F. K. Kan, A. B. Singh, and J. Liu. High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway

SREBP2 activation induces hepatic long-chain Acyl-CoA synthetase 1 (ACSL1) expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter

Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript

CETP inhibitors downregulate hepatic LDL receptor and PCSK9 expression in vitro and in vivo through a SREBP2 dependent mechanism

Background: CETP inhibitors block the transfer of cholesteryl ester from HDL-C to VLDL-C and LDL-C, thereby raising HDL-C and lowering LDL-C. In this study, we explored the effect of CETP inhibitors on hepatic LDL receptor (LDLR) and PCSK9 expression and further elucidated the underlying regulatory mechanism.Results: We first examined the effect of anacetrapib (ANA) and dalcetrapib (DAL) on LDLR and PCSK9 expression in hepatic cells in vitro. ANA exhibited a dose-dependent inhibition on both LDLR and PCSK9 expression in CETP-positive HepG2 cells and human primary hepatocytes as well as CETP-negative mouse primary hepatocytes (MPH). Moreover, the induction of LDLR protein expression by rosuvastatin in MPH was blunted by cotreatment with ANA. In both HepG2 and MPH ANA treatment reduced the amount of mature form of SREBP2 (SREBP2-M). In vivo, oral administration of ANA to dyslipidemic C57BL/6J mice at a daily dose of 50 mg/kg for 1 week elevated serum total cholesterol by approximately 24.5% (p < 0.05%) and VLDL-C by 70% (p < 0.05%) with concomitant reductions of serum PCSK9 and liver LDLR/SREBP2-M protein. Finally, we examined the in vitro effect of two other strong CETP inhibitors evacetrapib and torcetrapib on LDLR/PCSK9 expression and observed a similar inhibitory effect as ANA in a concentration range of 1-10 mu M.Conclusion: Our study revealed an unexpected off-target effect of CETP inhibitors that reduce the mature form of SREBP2, leading to attenuated transcription of hepatic LDLR and PCSK9. This negative regulation of SREBP pathway by ANA manifested in mice where CETP activity was absent and affected serum cholesterol metabolism. Published by Elsevier Ireland Ltd

A novel posttranscriptional mechanism for dietary cholesterol-mediated suppression of liver LDL receptor expression

It is well-established that over-accumulation of dietary cholesterol in the liver inhibits sterol-regulatory element binding protein (SREBP)-mediated LDL receptor (LDLR) gene transcription leading to a reduced hepatic LDLR mRNA level in hypercholesterolemic animals. However, it is unknown whether elevated cholesterol levels can elicit a cellular response to increase LDLR mRNA turnover to further repress LDLR expression in liver tissue. In the current study, we examined the effect of a high cholesterol diet on the hepatic expression of LDLR mRNA binding proteins in three different animal models and in cultured hepatic cells. Our results demonstrate that high cholesterol feeding specifically elevates the hepatic expression of LDLR mRNA decay promoting factor heterogeneous nuclear ribonucleoprotein (HNRNP) D without affecting expressions of other LDLR mRNA binding proteins in vivo and in vitro. Employing the approach of adenovirus-mediated gene knockdown, we further show that depletion of HNRNPD in the liver results in a marked reduction of serum LDL-cholesterol and a substantial increase in liver LDLR expression in hyperlipidemic mice. Additional studies of gene knockdown in albumin-luciferase-untranslated region (UTR) transgenic mice provide strong evidence supporting the essential role of 3'UTR in HNRNPD-mediated LDLR mRNA degradation in liver tissue. Altogether, this work identifies a novel post-transcriptional regulatory mechanism by which dietary cholesterol inhibits liver LDLR expression via inducing HNRNPD to accelerate LDLR mRNA degradation